Methods of increasing muscle mass and decreasing fat mass

Abstract

The present invention relates to compositions and methods for altering body composition in an individual, wherein the alteration in body composition is both an increase in muscle mass and a decrease in fat mass. The present invention also relates to compositions and methods for decreasing fat mass in an individual. The compositions and methods also increase muscle volume and lean body mass in an individual. The present invention also relates to compositions comprising a GDF8 inhibitor and an activin A inhibitor, and the use of such compositions to treat diseases and conditions characterized by increased fat mass and / or decreased muscle volume.

Description

[0001] This application was filed as a PCT international patent application on March 1, 2019, and claims priority to U.S. Provisional Application No. 62 / 637,017, filed on March 1, 2018, the disclosure of which is incorporated herein by reference in its entirety.

[0002] sequence list

[0003] This application includes an electronic sequence list entitled “Sequence-Listing-40848-091WOU1”, which was generated on March 1, 2019, and is 288 kilobytes (KB) (295,202 bytes) in size. The contents of the Txt document “Sequence-Listing-40848-091USU1” are incorporated herein by reference. Technical Field

[0004] This invention relates to compositions and methods for altering the body composition of an individual. The compositions and methods also reduce the individual's fat mass. They also increase the individual's muscle mass and / or lean body mass. More specifically, this invention relates to compositions comprising GDF8 inhibitors and activin A inhibitors, and the use of such compositions in treating diseases and conditions characterized by increased fat mass and / or decreased muscle mass or lean body mass. Background of the Invention

[0006] Growth and differentiation factor-8 (GDF8, also known as myostatin) is a secreted ligand belonging to the transforming growth factor-β (TGF-β) superfamily of growth factors. GDF8 plays a crucial role in the development and maintenance of skeletal muscle, acting as a negative regulator of myogenesis and skeletal muscle mass. Mutations (including knockouts) in GDF8 translate into a phenotype primarily characterized by increased muscle mass, but can also consist of changes in muscle formation (more muscle fibers), muscle fiber composition (larger muscle fiber cross-sectional area), and an increased protein / DNA ratio.

[0007] Antibodies and treatments for GDF8 are disclosed, for example, in US 8,840,894. Anti-GDF8 antibodies are also mentioned, for example, in US patent numbers 6,096,506; 7,320,789; 7,261,893; 7,807,159; 7,888,486; 7,635,760; 7,632,499; US patent applications 2006 / 0263354; 2007 / 0178095; 2008 / 0299126; 2010 / 0166764; 2009 / 0148436; and international patent applications WO2004 / 037861; WO2007 / 047112; WO2010 / 070094.

[0008] Activins belong to the transforming growth factor-β (TGF-β) superfamily and play a wide range of biological roles in cell proliferation, differentiation, metabolism, homeostasis, apoptosis, immune response, and tissue repair. Activin A is a disulfide-linked homodimer (two β-A chains) that binds to and activates heteropolymeric complexes of type I (Act RI-A and Act RI-B) and type II (Act RII-A and Act RII-B) serine-threonine kinase receptors.

[0009] Antibodies against activin A and their uses are disclosed, for example, in US 8,309,082; 9,718,881; and international patent application publication WO2008 / 031061.

[0010] Compositions and treatment methods comprising anti-GDF8 antibody and anti-activin A antibody are disclosed, for example, in US8,871,209.

[0011] Obesity is a global problem affecting one-third of the world's population. In the United States, the average obesity rate exceeds 20%. The cost of obesity-related diseases is staggering, totaling $190.2 billion, approximately 21% of annual healthcare costs in the United States. Obesity is an epidemic characterized by chronic low-grade inflammation associated with abnormal (elevated) fat mass. In participants of the Framingham Heart Study, abdominal obesity was associated with cardiovascular disease (CVD) events after adjusting for clinical risk factors and overall obesity. Britton JACC 2013 62; 921. Abdominal visceral fat accumulation is positively correlated with the progression of coronary non-calcified plaques. Imai Atherosclerosis 2012. Because high fat mass is associated with serious conditions such as congestive heart failure, hypertension / elevated blood pressure, pulmonary embolism, osteoarthritis, lymphedema, gastroesophageal reflux disease, chronic renal failure, cancer, fatty liver disease, and even depression, therapies to reduce total fat and / or android fat mass in individuals remain in demand.

[0012] Invention Summary

[0013] In one aspect, the present invention relates to a method for altering an individual's body composition, namely increasing muscle mass and reducing fat mass, comprising administering to the individual a first composition comprising an effective amount of a GDF8 inhibitor and a second composition comprising an effective amount of an activator A inhibitor. In another aspect, the present invention relates to a method for inducing a reduction in fat mass in an individual, the method comprising administering to the individual an effective amount of a composition comprising a GDF8 inhibitor and an activator A inhibitor.

[0014] In another aspect, the present invention relates to a method for treating diseases or disorders characterized by or related to increased body fat mass, the method comprising administering to an individual in need an effective amount of a first composition comprising an effective amount of a GDF8 inhibitor and a second composition comprising an effective amount of an activator A inhibitor. In another aspect, the present invention relates to a method for treating diseases or disorders characterized by or related to increased body fat mass, the method comprising administering to an individual in need an effective amount of a composition comprising a GDF8 inhibitor and an activator A inhibitor. In one aspect, the present invention relates to a method for altering an individual's body composition, i.e., a method for increasing muscle mass and decreasing body fat mass, the method comprising administering to an individual an effective amount of a GDF8 inhibitor and an effective amount of an activator A inhibitor, wherein there is a minimal and / or insignificant change in total body weight. Therefore, in one aspect of the invention, an individual administering an effective amount of a GDF8 inhibitor and an effective amount of an activator A inhibitor will experience an increase in muscle mass while a decrease in body fat mass, resulting in minimal and / or insignificant changes in total body weight.

[0015] In one aspect, the present invention relates to the use of GDF8 inhibitors and / or activin A inhibitors in the preparation of medicaments for achieving a reduction in an individual's body fat mass. In another aspect, the present invention relates to the use of GDF8 inhibitors and / or activin A inhibitors in the preparation of medicaments for treating diseases or disorders in an individual associated with increased body fat mass.

[0016] In some implementations, a GDF8 inhibitor is provided in a method for treating a disease or disorder characterized by increased fat mass, wherein the method includes administering a GDF8 inhibitor and an activin A inhibitor to an individual.

[0017] In some implementations, activin A inhibitors are provided in methods for treating diseases or disorders characterized by increased fat mass, wherein the methods include administering an activin A inhibitor and a GDF8 inhibitor to an individual.

[0018] In some implementations, non-therapeutic methods for reducing fat mass in an individual are provided, the methods comprising administering an activin A inhibitor and a GDF8 inhibitor to the individual.

[0019] In one embodiment of the method according to the invention, an effective amount of the GDF8 inhibitor comprises a dosing regimen selected from at least 0.1 mg / kg to about 10 gm / kg, 1 mg / kg to about 1 gm / kg, and 10 mg / kg to 100 mg / kg. In another embodiment of the method according to the invention, an effective amount of the GDF8 inhibitor comprises a single-dose dosing regimen selected from about 0.01 to about 20 mg / kg body weight, about 0.1 to about 10 mg / kg body weight, and about 0.1 to about 5 mg / kg body weight.

[0020] In another embodiment of the method according to the invention, an effective amount of activin A inhibitor comprises a dosing regimen selected from at least 0.1 mg / kg to about 10 gm / kg, 1 mg / kg to about 1 gm / kg, and 10 mg / kg to 100 mg / kg. In another embodiment of the method according to the invention, an effective amount of activin A inhibitor comprises a single-dose dosing regimen selected from about 0.01 to about 20 mg / kg body weight, about 0.1 to about 10 mg / kg body weight, and about 0.1 to about 5 mg / kg body weight.

[0021] In one embodiment of the method according to the invention, the effective amount of the GDF8 inhibitor is 6 mg / kg body weight and the effective amount of the activin A inhibitor is 3 mg / kg body weight. In another embodiment of the method according to the invention, the effective amount of the GDF8 inhibitor is 6 mg / kg body weight and the effective amount of the activin A inhibitor is 10 mg / kg body weight.

[0022] In one embodiment of the method according to the invention, the first composition is formulated for intravenous, subcutaneous, or oral administration. In another embodiment of the method according to the invention, the second composition is formulated for intravenous, subcutaneous, or oral administration. In some embodiments of the method according to the invention, the first and second compositions are administered to an individual simultaneously or sequentially.

[0023] In one embodiment of the method according to the invention, the first and second compositions are combined into a third composition prior to administration. In another embodiment, the third composition is formulated for intravenous, subcutaneous, or oral administration.

[0024] In one embodiment, the method according to the invention further includes determining the total fat mass of the individual prior to application. In another embodiment, the method according to the invention further includes determining the total fat mass of the individual after application, and applying the first and second compositions until the individual's total fat mass is reduced by at least 2% to 8%, 2.5% to 6%, 3% to 4%, or at least 2.0%, at least 2.5%, at least 3.0%, or at least 3.5% or more.

[0025] In one embodiment, the method according to the invention further includes measuring the amount of male-type fat in the individual prior to application. In another embodiment, the method according to the invention further includes measuring the amount of male-type fat in the individual after application, and applying the first composition and the second composition until the amount of male-type fat in the individual is reduced by at least 2% to 8%, 2.5% to 6%, 3% to 4%, or at least 2.0%, at least 2.5%, at least 3.0%, or at least 3.5% or more.

[0026] In one embodiment, the method according to the invention further includes measuring the volume of subcutaneous adipose tissue in the individual prior to application. In another embodiment, the method according to the invention further includes measuring the volume of subcutaneous adipose tissue in the individual after application, and applying the first composition and the second composition until the individual's male-type adipose mass is reduced by at least 2% to 8%, 2.5% to 6%, 3% to 4%, or at least 2.0%, at least 2.5%, at least 3.0%, or at least 3.5% or more.

[0027] In some embodiments, a method is provided that includes administering an effective amount of a GDF8 inhibitor and an effective amount of an activator A inhibitor to an individual in need, wherein the GDF8 inhibitor and the activator A inhibitor are administered together for a period of 48 hours or less, 24 hours or less, 12 hours or less, 6 hours or less, 3 hours or less, or 1 hour or less.

[0028] In some implementations, methods are provided that include administering an effective amount of a GDF8 inhibitor and an effective amount of an activator A inhibitor to an individual in need, wherein the individual exhibits a reduction in total fat mass, male-type fat mass, and / or subcutaneous adipose tissue volume.

[0029] In some implementations, methods are provided that include administering an effective amount of a GDF8 inhibitor and an effective amount of an activator A inhibitor to an individual in need, wherein the individual exhibits a reduction in total fat mass, male-type fat mass, and / or subcutaneous adipose tissue volume 4 weeks or longer after administration, or 8 weeks or longer.

[0030] In some implementations, methods are provided that include administering an effective amount of a GDF8 inhibitor and an effective amount of an activator A inhibitor to an individual in need, wherein the individual exhibits a reduction in total fat mass, male-type fat mass, and / or subcutaneous adipose tissue volume, and wherein the individual does not exhibit a reduction in intramuscular adipose tissue volume in the thigh.

[0031] In some implementations, a method is provided that includes administering an effective amount of a GDF8 inhibitor and an effective amount of an activator A inhibitor to an individual in need, wherein the individual exhibits a reduction in total fat mass, male-type fat mass, and / or subcutaneous adipose tissue volume, wherein the individual does not exhibit a reduction in intramuscular adipose tissue volume of the thigh 4 weeks or longer after administration, or 8 weeks or longer.

[0032] In some implementations, a kit is provided, which includes a first container containing an effective amount of GDF8 inhibitor and a second container containing an effective amount of specific activin A inhibitor.

[0033] In some implementations, the GDF8 inhibitor is a separable antibody or its antigen-binding fragment that specifically binds to GDF8.

[0034] In one embodiment of the method of the present invention, the GDF8 inhibitor is an antibody that specifically binds to GDF8 or an antigen-binding fragment thereof. In another embodiment, the antibody or antigen-binding fragment that specifically binds to GDF8 comprises a heavy chain complementarity-determining region (HCDR) containing the heavy chain variable region (HCVR) of SEQ ID NO:360, and a light chain complementarity-determining region (LCDR) containing the light chain variable region (LCVR) of SEQ ID NO:368. In yet another embodiment, the antibody or antigen-binding fragment that specifically binds to GDF8 comprises three HCDRs and three LCDRs, wherein the HCDRs comprise SEQ ID NO:362, SEQ ID NO:364, and SEQ ID NO:366, and the LCDRs comprise SEQ ID NO:370, SEQ ID NO:372, and SEQ ID NO:374.

[0035] In some implementations, the activin A inhibitor is a separable antibody or its antigen-binding fragment that specifically binds to activin A.

[0036] In one embodiment of the method according to the invention, the activin A inhibitor is an antibody that specifically binds to activin A or an antigen-binding fragment thereof. In another embodiment, the antibody or antigen-binding fragment that specifically binds to activin A comprises heavy chain complementarity-determining regions (HCDRs) containing the heavy chain variable region (HCVR) of SEQ ID NO:553 and light chain complementarity-determining regions (LCDRs) containing the light chain variable region (LCVR) of SEQ ID NO:537. In yet another embodiment, the antibody or antigen-binding fragment that specifically binds to activin A comprises three HCDRs and three LCDRs, wherein the HCDRs comprise SEQ ID NO:555, SEQ ID NO:557 and SEQ ID NO:559, and the LCDRs comprise SEQ ID NO:539, SEQ ID NO:541 and SEQ ID NO:543.

[0037] In one embodiment of the method according to the invention, the effective dose of the activin A inhibitor is selected from 100% to 200% of the effective dose of the GDF8 inhibitor, 100% to 250% of the effective dose of the GDF8 inhibitor, 100% to 300% of the effective dose of the GDF8 inhibitor, and 100% to 400% of the effective dose of the GDF8 inhibitor by weight.

[0038] In another embodiment of the method according to the invention, the weight ratio of the effective dose of the activin A inhibitor to the effective dose of the GDF8 inhibitor is 10:1 to 1:10, 8:1 to 1:8, 6:1 to 1:6, 3:1 to 1:3, or about 2:1 to 1:2. In another embodiment of the method according to the invention, the weight ratio of the effective dose of the activin A inhibitor is about 1.5 to 2.0 times the amount of the GDF8 inhibitor by weight.

[0039] In one embodiment of the method according to the invention, the GDF8 inhibitor is a bispecific antibody or antigen-binding fragment thereof that specifically binds to GDF8 and also specifically binds to activin A. In another embodiment, the activin A inhibitor is a bispecific antibody or antigen-binding fragment thereof that specifically binds to activin A and also specifically binds to GDF8.

[0040] In one embodiment of the method according to the invention, the reduction in fat mass in an individual is a reduction in total fat mass as measured by DXA (dual-energy X-ray absorptiometry). In another embodiment of the method according to the invention, the reduction in fat mass in an individual is a reduction in male-type fat mass as measured by DXA (dual-energy X-ray absorptiometry).

[0041] In one embodiment of the method according to the invention, the reduction in fat mass in an individual is a reduction in the volume of subcutaneous adipose tissue as measured by MRI (magnetic resonance imaging).

[0042] In one embodiment of the method according to the invention, an individual experiences an increase in muscle volume. The muscle volume may be the volume of thigh muscle tissue, for example, measured by MRI. In some embodiments, the muscle volume may be the volume of thigh muscle tissue, for example, measured by MRI. In some embodiments, the thigh muscle volume may be the volume of thigh muscle tissue including intramuscular adipose tissue and large blood vessels, or the volume of thigh muscle tissue excluding intramuscular adipose tissue and large blood vessels, for example, measured by MRI.

[0043] In one embodiment of the method according to the invention, the individual experiences an increase in total lean body mass. Total lean body mass can be measured by DXA (dual X-ray absorptiometry).

[0044] In one embodiment of the method according to the invention, the individual experiences an increase in limb lean body mass. Limb lean body mass can be measured by DXA and calculated, for example, by the aLBM equation.

[0045] In one embodiment of the method according to the invention, the individual experiences a reduction in total body fat, for example, as determined by DXA.

[0046] In one embodiment of the method according to the invention, the individual experiences a reduction in male-type body fat, for example, as measured by DXA.

[0047] In one embodiment of the method according to the invention, an individual experiences a reduction in the volume of subcutaneous adipose tissue, for example, as measured by DXA.

[0048] In one embodiment of the method according to the invention, an individual experiences a reduction in the total amount of fat in the arms and legs, for example, as measured by DXA.

[0049] In one embodiment of the invention, the individual does not exhibit a reduction in the volume of intramuscular fat tissue in the thigh, for example, as determined by MRI.

[0050] In one embodiment of the invention, the individual does not exhibit a decrease in total bone mineral density (BMD), as measured, for example by DXA.

[0051] In one embodiment of the invention, the individual does not exhibit a decrease in total bone mineral content (BMC), as measured, for example by DXA.

[0052] In one embodiment of the invention, the individual exhibits an increase in total bone mineral content (BMC), for example, as determined by DXA.

[0053] In another embodiment of the method according to the invention, the individual does not have muscular dystrophy or other diseases.

[0054] In some embodiments, kits are provided for altering body composition, reducing fat mass, increasing lean body mass, or treating diseases or disorders characterized by or associated with increased fat mass. The kits include a first container and a second container, the first container containing a composition containing an effective amount of a GDF8 inhibitor, and the second container containing a second composition containing an effective amount of an activin A inhibitor.

[0055] In some embodiments, a GDF8 inhibitor is provided for preparing a first composition for use as a medicament in a kit for altering body composition, reducing fat mass, increasing lean body mass, or treating diseases or disorders characterized by or associated with increased fat mass, the kit further comprising a second composition containing an activin A inhibitor.

[0056] In some embodiments, an activator A inhibitor is provided for preparing a first composition for use as a medicament in a kit for altering body composition, reducing fat mass, increasing lean body mass, or treating diseases or disorders characterized by or associated with increased fat mass, the kit further comprising a second composition containing a GDF8 inhibitor.

[0057] In some embodiments, a GDF8 inhibitor is provided for preparing a first composition for a kit used to alter an individual's body composition, reduce fat mass, or increase lean body mass, the kit further comprising a second composition containing an activin A inhibitor.

[0058] In some embodiments, an activator A inhibitor is provided for preparing a first composition for a kit used to alter an individual's body composition, reduce fat mass, or increase lean body mass, the kit further comprising a second composition containing a GDF8 inhibitor.

[0059] In some implementations, a first composition comprising a GDF8 inhibitor is provided for altering body composition, reducing fat mass, increasing lean body mass, or treating a disease or disorder characterized by or associated with increased fat mass or decreased lean body mass in an individual, wherein the individual has received a second composition comprising an activin A inhibitor.

[0060] In some implementations, a first composition comprising an activin A inhibitor is provided for altering body composition, reducing fat mass, increasing lean body mass, or treating a disease or disorder characterized by or associated with increased fat mass or decreased lean body mass in an individual, wherein the individual has received a second composition comprising a GDF8 inhibitor.

[0061] In some embodiments, a first composition comprising a GDF8 inhibitor is provided in a method for altering body composition, reducing fat mass, increasing lean body mass, or treating a disease or disorder characterized by or associated with increased fat mass or decreased lean body mass, the method further comprising administering a second composition comprising an activin A inhibitor.

[0062] In some embodiments, a first composition comprising an activin A inhibitor is provided in a method for altering body composition, reducing fat mass, increasing lean body mass, or treating a disease or disorder characterized by or associated with increased fat mass or decreased lean body mass, the method further comprising administering a second composition comprising a GDF8 inhibitor.

[0063] In some embodiments, compositions comprising an activator A inhibitor and a GDF8 inhibitor are provided for altering body composition, reducing fat mass, increasing lean body mass, or treating or preventing diseases or disorders characterized by or associated with increased fat mass or decreased lean body mass.

[0064] Other embodiments of the invention will become apparent upon reading the following detailed description. Brief description of the attached diagram

[0066] Figure 1 The bar chart depicts the clinical study results after 12 weeks in patients aged 70 years or older with sarcopenia who received anti-GDF8 antibody REGN1033 alone, as the mean LS and SE of the percentage change in total lean body mass from baseline compared to placebo. Patients receiving REGN1033 showed a significant increase in total lean body mass at 12 weeks in each of the three dosing regimens compared to placebo (n = 65). Patients receiving 100 mg anti-GDF8 antibody REGN1033 Q4W SC showed a 1.66% difference in total lean body mass compared to placebo (n = 62, P = 0.0077). Patients receiving 300 mg anti-GDF8 Q4W SC showed a 1.78% difference in total lean body mass compared to placebo (n = 64, P = 0.0043). Patients receiving 300 mg Q2W SC showed a 2.29% difference in total lean body mass compared to placebo (n = 59, P = 0.0004).

[0067] Figure 2A Table 1 shows the escalating dose groups for a study of 48 healthy postmenopausal women according to Example 2. A single intravenous dose of anti-GDF8 antibody REGN1033 and / or anti-activin A antibody REGN2477 was used. In the preliminary analysis, the placebo and high-dose combinations in each group were combined to obtain 12 individuals receiving placebo and 12 individuals receiving the high-dose combination, as shown in the boxed areas in the table.

[0068] Figure 2B The bar chart depicts the percentage change in thigh muscle volume (measured by MRI) at week 8 following a single intravenous administration of anti-activin A antibody REGN2477 and / or anti-GDF8 antibody REGN1033 in healthy postmenopausal women. The anti-GDF8 (6 mg / kg), anti-GDF8 (6 mg / kg) + medium-dose anti-activin A (3 mg / kg), and anti-GDF8 (6 mg / kg) + high-dose anti-activin A (10 mg / kg) groups showed a significant increase in the percentage change in thigh muscle volume compared to placebo. (*Nominal P < 0.5, relative to placebo; ****Nominal P < 0.0001, relative to placebo).

[0069] Figure 2C The bar chart depicts the change in total fat mass (as determined by DXA) % at week 8 following a single intravenous administration of anti-activin A antibody REGN2477 and / or anti-GDF8 antibody REGN1033 in healthy postmenopausal women. The figures show the change compared to placebo. The anti-GDF8 (6 mg / kg) + high-dose anti-activin A (10 mg / kg) group showed a significant reduction in the change in total fat mass % (*nominal P < 0.05, relative to placebo).

[0070] Figure 3 The diagram shows a line graph depicting the percentage change (by MRI) in mean LS (SE) thigh muscle volume from baseline at weeks 0, 4, and 8 following intravenous administration of anti-activin A antibody REGN2477 and / or anti-GDF8 antibody REGN1033 in healthy postmenopausal women. After a single intravenous dose for 4 weeks, each of the following groups—anti-GDF8 (6 mg / kg), anti-GDF8 (6 mg / kg) + low-dose anti-activin A (1 mg / kg), anti-GDF8 (6 mg / kg) + medium-dose anti-activin A (3 mg / kg), and anti-GDF8 (6 mg / kg) + high-dose anti-activin A (10 mg / kg)—showed a significant increase in the percentage change in thigh muscle volume (measured by MRI, excluding intramuscular adipose tissue) compared to placebo. Eight weeks after a single intravenous dose, significant increases in thigh muscle volume percentage (%) relative to placebo were observed in both the anti-GDF8 (6 mg / kg) + medium-dose anti-activin A (3 mg / kg) and anti-GDF8 (6 mg / kg) + high-dose anti-activin A (10 mg / kg) groups. (*Nominal P < 0.05, **Nominal P < 0.001). N values ​​for each group are shown in [Figure / Table / Insert N value here]. Figure 2A middle.

[0071] Figure 4 Line graphs are shown depicting individual data (by MRI) at weeks 0, 4, and 8 following a single intravenous administration of the anti-activin A antibody REGN2477 and / or the anti-GDF8 antibody REGN1033 to healthy postmenopausal women in multiple individual groups: placebo, anti-GDF8 (6 mg / kg), high-dose anti-activin A (10 mg / kg), anti-GDF8 (6 mg / kg) + low-dose anti-activin A (1 mg / kg), anti-GDF8 (6 mg / kg) + medium-dose anti-activin A (3 mg / kg), and anti-GDF8 (6 mg / kg) + high-dose anti-activin A (10 mg / kg). The percentage change is expressed as a % change relative to baseline. An increase in thigh muscle volume was consistently observed in all individuals treated with the REGN2477 + REGN1033 combination. Different lines within each treatment group represent different individuals.

[0072] Figure 5The graph shows the mean (least squared) LS (least squared) change from baseline in limb lean (body) mass (i.e., the sum of lean tissue in the arms and legs) at weeks 0, 4, and 8 following a single intravenous administration of anti-activin A antibody REGN2477 and / or anti-GDF8 antibody REGN1033 in six groups of healthy postmenopausal women. The N value for each group is shown in the graph. Figure 2A At 4 and 8 weeks, each of the three combination dose groups, including anti-GDF8 (6 mg / kg) + low-dose anti-activin A (1 mg / kg), anti-GDF8 (6 mg / kg) + medium-dose anti-activin A (3 mg / kg), and anti-GDF8 (6 mg / kg) + high-dose anti-activin A (10 mg / kg), showed a significant increase in % change in limb lean body mass compared to placebo (LS mean difference *nominal p < 0.05, **nominal p < 0.001). N values ​​for each group are shown in [figure missing]. Figure 2A middle.

[0073] Figure 6 Line graphs are shown depicting the changes in mean (SE = standard error) percentage of total fat mass (as determined by DXA) at weeks 0, 4, and 8 in healthy postmenopausal women following a single intravenous administration of anti-activin A antibody REGN2477 and / or anti-GDF8 antibody REGN1033 to the placebo, anti-GDF8 (6 mg / kg), high-dose anti-activin A (10 mg / kg), anti-GDF8 (6 mg / kg) + low-dose anti-activin A (1 mg / kg), anti-GDF8 (6 mg / kg) + medium-dose anti-activin A (3 mg / kg), and anti-GDF8 (6 mg / kg) + high-dose anti-activin A (10 mg / kg) groups. N values ​​for each group are shown in the figure. Figure 2A In the high-dose group (anti-GDF8 (6 mg / kg) + high-dose anti-activin A (10 mg / kg)), the difference in percentage change (LS mean) compared with placebo at weeks 4 and 8 showed a significant reduction in total fat mass (*nominal P < 0.05). Blockade of activin A and GDF8 resulted in a reduction in total fat mass, as assessed by DXA.

[0074] Figure 7A line graph is shown depicting the mean (SE) percentage change in male-type body fat mass at weeks 0, 4, and 8 following a single intravenous administration of anti-activin A antibody REGN2477 and / or anti-GDF8 antibody REGN1033 in healthy postmenopausal women across six groups: placebo, anti-GDF8 (6 mg / kg), high-dose (10 mg / kg) anti-activin A, anti-GDF8 (6 mg / kg) + low-dose (1 mg / kg) anti-activin A, anti-GDF8 (6 mg / kg) + medium-dose (3 mg / kg) anti-activin A, and anti-GDF8 (6 mg / kg) + high-dose (10 mg / kg) anti-activin A. Compared to placebo, the high-dose REGN1033 + REGN2477 group showed a significant difference in the mean LS percentage change in male-type body fat mass (by DXA) at weeks 4 and 8 (*nominal P < 0.05). The N values ​​for each group are shown below. Figure 2A In the study, as assessed by DXA, the blockade of activin A and GDF8 was also associated with a decrease in male-type body fat.

[0075] Figure 8 A line graph is shown depicting the mean percentage change of the sum of colors (LS) with SE in thigh muscle volume (excluding intramuscular adipose tissue and large vessels) at weeks 0, 4, and 8 following a single intravenous administration of anti-activin A antibody REGN2477 and / or anti-GDF8 antibody REGN1033 in healthy postmenopausal women. The six groups included placebo, anti-GDF8 (6 mg / kg), high-dose (10 mg / kg) anti-activin A, anti-GDF8 (6 mg / kg) + low-dose (1 mg / kg) anti-activin A, anti-GDF8 (6 mg / kg) + medium-dose (3 mg / kg) anti-activin A, and anti-GDF8 (6 mg / kg) + high-dose (10 mg / kg) anti-activin A. The N values ​​for each group are shown in [the graph]. Figure 2A Compared with placebo, at weeks 4 and 8, the REGN2477+REGN1033 medium and high groups showed a significant increase in mean percentage change in thigh muscle volume. (*Nominal P < 0.05, **Nominal P < 0.001).

[0076] Figure 9A line graph is shown depicting the mean percentage change in total lean body mass (LS) with SE at weeks 0, 4, and 8 following a single intravenous administration of anti-activin A antibody REGN2477 and / or anti-GDF8 antibody REGN1033 to six groups of healthy postmenopausal women. These six groups included placebo, anti-GDF8 (6 mg / kg), high-dose (10 mg / kg) anti-activin A, anti-GDF8 (6 mg / kg) + low-dose (1 mg / kg) anti-activin A, anti-GDF8 (6 mg / kg) + medium-dose (3 mg / kg) anti-activin A, and anti-GDF8 (6 mg / kg) + high-dose (10 mg / kg) anti-activin A. The N value for each group is shown in [the graph]. Figure 2A middle.

[0077] Compared with placebo, at 4 and 8 weeks, the medium-dose and high-dose groups of REGN2477+REGN1033 showed a significant increase in the change in total lean body mass percentage compared with placebo. (*Nominal P < 0.05).

[0078] Figure 10 A line graph is shown depicting the mean percentage change in LS (less than SE) of limb lean body mass (calculated by the aLBM equation) (kg) at weeks 0, 4, and 8 following a single intravenous administration of anti-activin A antibody REGN2477 and / or anti-GDF8 antibody REGN1033 to healthy postmenopausal women in six groups: placebo, anti-GDF8, high-dose (10 mg / kg) anti-activin A, anti-GDF8 + low-dose (1 mg / kg) anti-activin A, anti-GDF8 + medium-dose (3 mg / kg) anti-activin A, and anti-GDF8 + high-dose (10 mg / kg) anti-activin A. The N value for each group is shown in... Figure 2A Compared with placebo, at 4 and 8 weeks, in each of the low, medium and high dose groups, REGN2477+REGN1033 treatment resulted in a significant increase in the percentage change of limb lean body mass (kg) calculated by the aLBM equation (*p<0.05, **p<0.001).

[0079] Figure 11A line graph is shown depicting the mean percentage change in total fat mass (kg) with SE at weeks 0, 4, and 8 following a single intravenous administration of anti-activin A antibody REGN2477 and / or anti-GDF8 antibody REGN1033 to six groups of healthy postmenopausal women. These six groups included placebo, anti-GDF8 (6 mg / kg), high-dose (10 mg / kg) anti-activin A, anti-GDF8 + low-dose (1 mg / kg) anti-activin A, anti-GDF8 (6 mg / kg) + medium-dose (3 mg / kg) anti-activin A, and anti-GDF8 (6 mg / kg) + high-dose (10 mg / kg) anti-activin A. The N values ​​for each group are shown in [the graph]. Figure 2A The high-dose combination of REGN2477 and REGN1033 showed a significant reduction in total fat mass % at 8 weeks: -3.92% compared with placebo (-0.65%) (high-dose group). (*Nominal P < 0.05).

[0080] Figure 12 The graph shows the thigh muscle volume (cm²) at weeks 0, 4, and 8 following a single intravenous administration of anti-activin A antibody REGN2477 and / or anti-GDF8 antibody REGN1033 to healthy postmenopausal women in six groups. 3 The mean percentage change of LS with SE in (including intramuscular adipose tissue and large blood vessels) was defined in six groups: placebo, anti-GDF8, high-dose (10 mg / kg) anti-activin A, anti-GDF8 + low-dose (1 mg / kg) anti-activin A, anti-GDF8 + medium-dose (3 mg / kg) anti-activin A, and anti-GDF8 + high-dose (10 mg / kg) anti-activin A. The N value for each group is shown in [the table / image ... Figure 2A Compared with placebo, in the high and medium dose treatment groups at 4 and 8 weeks, the high and medium dose REGN2477+REGN1033 treatment groups showed a significant increase in the percentage change of thigh muscle volume (including intramuscular adipose tissue and large blood vessels) (*nominal p<0.05, **nominal p<0.001).

[0081] Figure 13A line graph is shown, depicting the mean percentage change in LS (left limb mass, the sum of lean body mass in arms and legs) with SE at weeks 0, 4, and 8 following a single intravenous administration of anti-activin A antibody REGN2477 and / or anti-GDF8 antibody REGN1033 to healthy postmenopausal women in six groups: placebo, anti-GDF8, high-dose (10 mg / kg) anti-activin A, anti-GDF8 + low-dose (1 mg / kg) anti-activin A, anti-GDF8 + medium-dose (3 mg / kg) anti-activin A, and anti-GDF8 + high-dose (10 mg / kg) anti-activin A. The N value for each group is shown in... Figure 2A In the mean percentage change of limb lean body mass relative to placebo was significantly increased in each of the REGN2477+REGN1033 low, medium and high treatment groups at 4 and 8 weeks (*nominal p<0.05, *nominal *p<0.001).

[0082] Figure 14 A line graph is shown depicting the mean percentage change in the LS (lower serous body mass) with SE in male-type fat mass (kg) at weeks 0, 4, and 8 following a single intravenous administration of anti-activin A antibody REGN2477 and / or anti-GDF8 antibody REGN1033 in six groups of healthy postmenopausal women: placebo, anti-GDF8, high-dose (10 mg / kg) anti-activin A, anti-GDF8 + low-dose (1 mg / kg) anti-activin A, anti-GDF8 + medium-dose (3 mg / kg) anti-activin A, and anti-GDF8 + high-dose (10 mg / kg) anti-activin A. The N value for each group is shown in... Figure 2A In contrast to placebo, the high-dose REGN1033+REGN2477 group showed a significantly lower percentage change in male-type body fat mass (via DXA) at weeks 4 and 8 (*nominal p<0.05).

[0083] Figure 15 The figure shows a line graph depicting the volume of intramuscular adipose tissue in the thigh at weeks 0, 4, and 8 following a single intravenous administration of anti-activin A antibody REGN2477 and / or anti-GDF8 antibody REGN1033 in six groups of healthy postmenopausal women. 3 The mean percentage change of LS with SE, the six groups including placebo, anti-GDF8, high-dose (10 mg / kg) anti-activin A, anti-GDF8 + low-dose (1 mg / kg) anti-activin A, anti-GDF8 + medium-dose (3 mg / kg) anti-activin A, and anti-GDF8 + high-dose (10 mg / kg) anti-activin A. The N value for each group is shown in Figure 2AIn the 8-week period, the high-dose REGN1033+REGN2477 group showed an increase in intramuscular fat volume in the thigh compared with placebo (as mean % change). (*Nominal p<0.05).

[0084] Figure 16 A line graph is shown depicting the mean percentage change in LS (with SE) of total intramuscular and perimuscular adipose tissue (IMAT) at weeks 0, 4, and 8 following a single intravenous administration of anti-activin A antibody REGN2477 and / or anti-GDF8 antibody REGN1033 to six groups of healthy postmenopausal women, including placebo, anti-GDF8, high-dose (10 mg / kg) anti-activin A, anti-GDF8 + low-dose (1 mg / kg) anti-activin A, anti-GDF8 + medium-dose (3 mg / kg) anti-activin A, and anti-GDF8 + high-dose (10 mg / kg) anti-activin A. The N value for each group is shown in... Figure 2A In weeks 4 and 8, there was no significant difference between the REGN1033+REGN2477 combination treatment group and placebo in the mean percentage change of LS with SE in the total intramuscular and perimuscular adipose tissue (IMAT).

[0085] Figure 17 The figure shows a line graph depicting subcutaneous adipose tissue volume (cm³) at weeks 0, 4, and 8 following a single intravenous administration of anti-activin A antibody REGN2477 and / or anti-GDF8 antibody REGN1033 to six groups of healthy postmenopausal women. 3 The mean percentage change of LS with SE, the six groups including placebo, anti-GDF8, high-dose (10 mg / kg) anti-activin A, anti-GDF8 + low-dose (1 mg / kg) anti-activin A, anti-GDF8 + medium-dose (3 mg / kg) anti-activin A, and anti-GDF8 + high-dose (10 mg / kg) anti-activin A. The N value for each group is shown in Figure 2A In the 8-week study, compared with placebo, the low and medium treatment groups of REGN1033+REGN2477 showed a significant reduction in the percentage change in subcutaneous adipose tissue volume (*nominal p<0.05). Invention Details

[0087] Before describing this invention, it should be understood that the invention is not limited to the specific compositions, methods, and experimental conditions described, as these compositions, methods, and conditions can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the invention is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0088] Unless the context clearly specifies otherwise, the singular form used in this specification and the appended claims includes the plural indicator. Thus, for example, reference to "method" includes one or more methods and / or steps of the type described herein, and / or others that will become apparent to those skilled in the art upon reading this disclosure.

[0089] As used herein, when referring to a particular enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated value. For example, as used herein, the expression “about 100” includes 99 and 101, as well as all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0090] Although any methods and materials similar to or equivalent to those described herein may be used to practice the invention, preferred methods and materials are described hereafter. All publications mentioned herein are incorporated herein by reference in their entirety.

[0091] Antibodies and antigen-binding fragments of antibodies

[0092] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule comprising four polypeptide chains (two heavy (H) chains and two light (L) chains) linked together by disulfide bonds, as well as its multimers (e.g., IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or V). H The heavy-chain constant region contains three structural domains, C H 1. C H 2 and C H 3. Each light chain contains a light chain variable region (abbreviated as LCVR or V in this document). L The light chain constant region contains a structural domain (C) and a light chain constant region. L 1). V H and V L The region can be further subdivided into highly variable regions called complementary determinant regions (CDRs), interspersed with more conservative regions called framing regions (FRs). Each V H and V L It comprises three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the antibody (or its antigen-binding portion) may be identical to the human germline sequence, or may be natural or artificially modified. The common amino acid sequence may be defined based on side-by-side analysis of two or more CDRs.

[0093] Antibodies may be referred to herein using the following nomenclature: an Fc prefix (e.g., "H1M," "H2aM," "H4H") followed by a numerical identifier (e.g., "10446"), and then a suffix of "P," "P2," or "N." Thus, according to this nomenclature, an antibody may be referred to herein as, for example, "H4H10446P2." The H1M, H2M, and H4H prefixes in antibody names used herein indicate a specific Fc region isotype of the antibody. For example, an "H2aM" antibody has mouse IgG2a Fc, while an "H4H" antibody has human IgG4 Fc. As will be understood by those skilled in the art, antibodies with a specific Fc isotype can be converted into antibodies with different Fc isotypes (e.g., an antibody with mouse IgG2a Fc can be converted into an antibody with human IgG4, etc.), but in any case, the variable regions (including CDRs) will remain the same, and the expected binding properties will be the same or substantially similar, regardless of the nature of the Fc region.

[0094] As used herein, the term "antibody" also includes the antigen-binding fragment of a complete antibody molecule. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be obtained from, for example, complete antibody molecules using any suitable standard technique such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding variable and optionally constant domains of the antibody. Such DNA is known and / or readily available from, for example, commercially available sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques to, for example, align one or more variable and / or constant domains to a suitable conformation, or introduce codons, generate cysteine ​​residues, modify, add or delete amino acids, etc.

[0095] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) the smallest recognition unit consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., a separated complementarity-determining region (CDR), such as a CDR3 peptide) or a restricted FR3-CDR3-FR4 peptide. As used herein, the expression "antigen-binding fragment" also includes other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-transplanted antibodies, biantibodies, triantibodies, tetraantibodies, microantibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains.

[0096] Antibody antigen-binding fragments typically contain at least one variable domain. The variable domain can be of any size or have any amino acid composition, and will typically contain at least one CDR adjacent to or conforming to one or more frame sequences. L In the antigen-binding fragment of the VH domain associated with the domain, V H Domain and V L Domains can be positioned relative to each other in any suitable arrangement. For example, a variable region can be dimerized and contain V. H -V H V H -V L or V L -V L Dimer. Optionally, the antigen-binding fragment of the antibody may contain monomer V. H Domain or V L Structural domain.

[0097] In some embodiments, the antigen-binding fragment of the antibody may include at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant regions that may be found within the antigen-binding fragment of the antibody used in this invention include: (i) V H -C H 1; (ii)V H -C H 2; (iii)V H -C H 3; (iv)V H -C H 1-C H 2; (v)V H -C H 1-C H 2-CH 3;(vi)V H -C H 2-C H 3;(vii)V H -C L (viii)V L -C H 1; (ix)V L -C H 2; (x)V L -C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3; (xiii)V L -C H 2-C H 3; and (xiv)V L -C L In any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly connected to each other or may be connected by complete or partial hinge regions or linker regions. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, creating a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single peptide molecule. Furthermore, the antigen-binding fragment of the antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) non-covalently associated with each other and / or associated with one or more monomeric VH or VL domains (e.g., via disulfide bonds).

[0098] Intact antibody molecules and antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antibody or antigen-binding fragments typically contain at least two distinct variable domains, each capable of specifically binding to a single antigen or different epitopes on the same antigen. Using conventional techniques available in the art, any form of multispecific antibody can be adapted to the antibody or antigen-binding fragments described herein.

[0099] The antibodies used in the compositions and methods of the present invention can act via complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). “Complement-dependent cytotoxicity” (CDC) refers to the lysis of cells expressing antigens by the antibodies of the present invention in the presence of complement. “Antibody-dependent cell-mediated cytotoxicity” (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies bound to target cells, thereby causing target cell lysis. CDC and ADCC can be measured using assays well known and available in the art. (See, for example, U.S. Patents 5,500,362 and 5,821,337, and Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998)).

[0100] The antibodies described herein may comprise or consist of human antibodies and / or recombinant human antibodies or fragments thereof, or be composed of human antibodies and / or recombinant human antibodies or fragments thereof. As used herein, the term "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may also include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo), such as in CDRs, particularly in CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from other mammalian species (e.g., mice) have been grafted onto human frame sequences.

[0101] The antibody molecules described herein may comprise or consist of recombinant human antibodies or antigen-binding fragments thereof. The term "recombinant human antibody" as used herein is intended to include all human antibodies prepared, expressed, generated, or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into host cells, antibodies isolated from recombinant or combined human antibody libraries, antibodies isolated from animals (e.g., mice) transgenic with human immunoglobulin genes (see, for example, Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other method involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies are mutagenized in vitro (or, when using transgenic animals with human Ig sequences, in vivo somatic cell mutagenization), thus reducing the V of the recombinant antibody. H and V L The amino acid sequence of the region is such that, although it originates from human lineage V,H and V L The sequence is related to, but may not be naturally present in all components of the human antibody lineage in vivo. Recombinant antibodies can be used in other embodiments of the compositions and methods of the present invention.

[0102] As used herein, "isolated antibody" means an antibody that has been identified and isolated and / or recovered from at least one component of its natural environment. For example, an antibody isolated or removed from at least one component of an organism, tissue, or cell (in which the antibody is naturally present or naturally produced) is an "isolated antibody" for the purposes of this invention. Isolated antibodies also include recombinant intracellular in situ antibodies, as well as antibodies that have undergone at least one purification or isolation step. According to some embodiments, isolated antibodies may be substantially free of other cellular material and / or chemicals. Isolated antibodies can be used in further embodiments of the compositions and methods of this invention.

[0103] A protein or polypeptide is considered "substantially pure," "substantially homogeneous," or "substantially purified" when at least about 60 to 75% of the sample shows a single type of polypeptide. The polypeptide or protein can be a monomer or a polymer. A substantially pure polypeptide or protein typically comprises about 50%, 60, 70%, 80%, or 90% w / w of the protein sample, typically about 95%, preferably more than 99% pure. Protein purity or homogeneity can be indicated by a variety of methods well known in the art, such as polyacrylamide gel electrophoresis of the protein sample followed by observation of individual polypeptide bands after staining the gel with a staining agent well known in the art. For certain purposes, higher resolution can be provided by using HPLC or other purification methods well known in the art.

[0104] As used herein, the term "peptide analog or variant" refers to a polypeptide consisting of a segment of at least 25 amino acids that is substantially identical to a portion of an amino acid sequence and has at least one of the following properties: (1) specifically binding to GDF8 under suitable binding conditions, or (2) the ability to block the biological activity of GDF8. Typically, peptide analogs or variants contain conserved amino acid substitutions (or insertions or deletions) relative to the naturally occurring sequence. Analogs are typically at least 20 amino acids long, at least 50, 60, 70, 80, 90, 100, 150, or 200 amino acids long or longer, and can generally be as long as the full-length naturally occurring polypeptide.

[0105] Preferred amino acid substitutions are those that: (1) reduce sensitivity to proteolysis; (2) reduce sensitivity to oxidation; (3) alter the binding affinity for forming the protein complex; (4) alter the binding affinity; and (5) impart or alter other physicochemical or functional properties to these analogs. Analogs may include various mutations that differ from naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (preferably conserved amino acid substitutions) may be made in naturally occurring sequences (preferably in portions of the polypeptide outside the domains forming intermolecular contacts). Conserved amino acid substitutions should not substantially alter the structural characterization of the parent sequence (e.g., substitutions should not tend to disrupt helices present in the parent sequence or disrupt other types of secondary structures characterizing the parent sequence). Examples of recognized peptide secondary and tertiary structures are described in *Principles of Protein, Structure and Molecular Biology* (Creighton, 1984; W.H. Freeman and Company, New York; *Introduction to Protein Structure* (Branden & Tooze, eds., 1991, Garland Publishing, NY); and Thornton et al., 1991, *Nature* 354:105, each incorporated herein by reference.

[0106] Non-peptide analogs are commonly used in the pharmaceutical industry as drugs that have properties similar to those of the template peptide. These types of non-peptide compounds are referred to as "peptide mimics" or "mimic peptides" (see, for example, Fauchere (1986) J. Adv. Drug Res. 15:29; and Evans et al. (1987) J. Med. Chem. 30:1229, which are incorporated herein by reference). More stable peptides can also be produced by substituting one or more amino acids of the common sequence with the same type of D-amino acid system (e.g., D-lysine instead of L-lysine). Furthermore, bound peptides containing the common sequence or substantially the same common sequence variation can be produced by methods known in the art (Rizo et al. (1992) Ann. Rev. Biochem. 61:387, which are incorporated herein by reference) through the addition of an internal cysteine ​​residue capable of forming an intramolecular disulfide bond of a cyclized peptide.

[0107] When applied to peptides, the terms "substantial identity" or "substantially identical" mean that, when optimally aligned using procedures such as GAP or BESTFIT with default gap weights, two peptide sequences share at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity. Preferably, the difference in the position of the dissimilar residues lies in conserved amino acid substitutions. A "conserved amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by another amino acid residue in a side chain (R group) having similar chemical properties (e.g., charge or hydrophobicity). Typically, conserved amino acid substitutions do not substantially alter the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conserved substitutions, the percentage of sequence identity or degree of similarity can be upregulated to correct for the conservatism of the substitution. The means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of amino acid groups with side chains having similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; and 6) sulfur-containing side chains: cysteine ​​and methionine. Preferred groups of conserved amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Optionally, "conservative" is replaced by any change that has a positive value in the PAM250 log-likelihood matrix as disclosed in Gonnet et al. (1992) Science 256:1443-45 (incorporated hereby by reference). "Moderately conservative" is replaced by any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0108] Sequence similarity (also known as sequence identity) of peptides is typically determined using sequence analysis software. Protein analysis software uses similarity measures assigned to multiple substitutions, deletions, and other modifications, including conserved amino acid substitutions, to match similar sequences. For example, GCG includes programs such as “Gap” and “Bestfit”, which can determine sequence homology or sequence identity between closely related peptides (such as homologous peptides from different species, or wild-type proteins and their mutants) using default parameters. See, for example, GCG version 6.1. Peptide sequences can also be compared using the program FASTA in GCG version 6.1 using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides an alignment between the query sequence and the search sequence and a percentage of sequence identity for the best overlapping region (Pearson (2000), ibid.). Another preferred algorithm when comparing the sequences of the present invention with a database containing a large number of sequences from different organisms is the computer program BLAST, specifically blastp or tblastn (using default parameters). See, for example, Altschul et al. (1990) J.Mol.Biol. 215: 403-410 and Altschul et al. (1997) NucleicAcids Res. 25: 3389-402, each of which is incorporated herein by reference.

[0109] The length of polypeptide sequences compared for homology is typically at least about 16 amino acid residues, at least about 20 residues, at least about 24 residues, at least about 28 residues, or at least about 35 residues. Comparing amino acid sequences is preferred when searching databases containing sequences from a large number of different organisms.

[0110] In some embodiments, the present invention relates to a method for altering the composition of an individual's body, the method comprising administering to the individual a first composition comprising an effective amount of a GDF8 inhibitor and a second composition comprising an effective amount of an activator A inhibitor.

[0111] In some embodiments, the present invention relates to a method for inducing a reduction in an individual's body fat mass, the method comprising administering to the individual a first composition containing an effective amount of a GDF8 inhibitor and a second composition containing an effective amount of an activin A inhibitor.

[0112] In some embodiments, the present invention relates to a method for inducing an increase in muscle mass in an individual, the method comprising administering to an individual in need a first composition comprising an effective amount of a GDF8 inhibitor and a second composition comprising an effective amount of an activator A inhibitor.

[0113] In some embodiments, the present invention relates to a method for altering the composition of an individual's body, the method comprising administering to the individual a composition comprising an effective amount of a GDF8 inhibitor and an effective amount of an activator A inhibitor.

[0114] In some embodiments, the present invention relates to a method for inducing a reduction in an individual's body fat mass, the method comprising administering to the individual a composition comprising an effective amount of a GDF8 inhibitor and an effective amount of an activator A inhibitor.

[0115] In some embodiments, the present invention relates to a method for inducing an increase in muscle mass in an individual, the method comprising administering to an individual in need a composition comprising an effective amount of a GDF8 inhibitor and an effective amount of an activator A inhibitor.

[0116] In some implementations, the individual is a human individual. The human individual can be an adult human individual. The individual can be male or female. The individual can be healthy. The individual may have an undesirable amount of body fat. The individual may have a disease or condition characterized by increased body fat and / or decreased muscle mass or lean body mass, or be at risk of having said disease or condition. The individual can be a postmenopausal female individual. The individual can be a male or female individual aged 40 or older, 50 or older, 60 or older, or 70 or older.

[0117] In some embodiments, the present invention relates to a method comprising administering to an individual in need a composition comprising a GDF8 inhibitor and a composition comprising an activator A inhibitor for the treatment or prevention of diseases and conditions characterized by increased fat mass and / or decreased muscle mass or lean body mass.

[0118] In some embodiments of the invention, an individual suffers from at least one disease or disorder that may be associated with increased fat mass. In some embodiments, said disease or disorder may be selected from obesity, metabolic syndrome, nutritional disorders, high cholesterol, dyslipidemia, cardiovascular disease, cellulitis, cancer (including colon cancer, esophageal cancer, kidney cancer, pancreatic cancer, gallbladder cancer, breast cancer, or endometrial cancer), polycystic ovary syndrome, gout, gallbladder disease, sleep apnea, respiratory disorders, asthma, osteoarthritis, cataracts, congestive heart failure, cardiomegaly, hypertension / elevation of blood pressure, pulmonary embolism, lymphedema, gastroesophageal reflux disease, hernia, chronic renal failure, urinary incontinence, connective tissue diseases, and fatty liver disease. In another embodiment, said disease or disorder may be sarcopenia.

[0119] GDF8 inhibitors

[0120] The present invention includes methods for altering an individual's body composition, inducing a reduction in an individual's fat mass and increasing an individual's lean body mass, and methods for treating diseases or disorders characterized by increased fat mass in an individual, said methods including administering to the individual a composition containing an effective amount of a GDF8 inhibitor.

[0121] The term "GDF8" (also known as "growth and differentiation factor-8" and "myogenic inhibitory protein") refers to a protein (mature protein) having the amino acid sequence SEQ ID NO: 340. According to the present invention, a GDF8-specific binding protein specifically binds to GDF8 but not to other ActRIIB ligands, such as GDF3, BMP2, BMP4, BMP7, BMP9, BMP10, GDF11, activin A, activin B, activin AB, Nodal, etc.

[0122] As used herein, "GDF8 inhibitor" is any substance that binds to or interacts with human GDF8 and interferes with or inhibits the normal biological function of GDF8 in vitro or in vivo. Non-limiting examples of the GDF8 inhibitor class include small molecule GDF8 antagonists, nucleic acid-based inhibitors of GDF8 expression or activity (e.g., siRNA or antisense), peptide-based molecules that specifically interact with GDF8 (e.g., peptide antibodies), receptor molecules that specifically interact with GDF8, GDF8-binding scaffold molecules, proteins comprising a ligand-binding portion of a receptor that specifically binds to GDF8, and anti-GDF8 aptamers or portions thereof. In a preferred embodiment, the GDF8 inhibitor used in the context of this invention is an anti-GDF8 antibody or an antigen-binding fragment thereof that specifically binds to human GDF8. Anti-GDF8 antibodies include neutralizing and / or blocking antibodies. Inhibition caused by anti-GDF8 neutralizing and / or blocking antibodies need not be complete, as long as it can be detected by appropriate assays.

[0123] As used herein, the term "anti-GDF8 antibody" also includes multispecific antigen-binding molecules (e.g., bispecific antibodies) wherein at least one binding domain (e.g., "binding arm") of the multispecific antigen-binding molecule specifically binds to GDF8.

[0124] Exemplary anti-GDF8 antibodies that can be used in the compositions and methods of the present invention include, for example, fully human anti-GDF8 antibody H4H1657N2, also known as REGN1033, (e.g., an anti-GDF8 antibody comprising heavy and light chain variable regions having amino acid sequences SEQ ID NO:360 and SEQ ID NO:368, respectively, as described in U.S. Patent No. 8,840,894).Other GDF8 antagonists that can be used in the compositions and methods of the present invention include anti-GDF8 antibodies as described in US2006 / 0263354 and US7,807,159 (e.g., antibodies named 2_112_1, such as having the ATCC depositary name PTA-6574, or antibodies named 2_112_K, such as having the HCVR / LCVR amino acid sequence SEQ ID NO:620 and 621); anti-GDF8 antibodies as described in US Patent 8,999,343 and US Publication 2013 / 0209489 (e.g., 12A5-5, such as having the HCVR / LCVR amino acid sequence SEQ ID NO:622 and 623); and anti-GDF8 antibodies as described in US Publication 2013 / 0142788 (e.g., 10B3H8L5, such as having the HCVR / LCVR amino acid sequence SEQ ID NO:622 and 623). NO:624 and 625, and 10B3H8L5-Fc-inactivated); anti-GDF8 antibodies as described in U.S. Patents 8,940,874 and 7,261,893 (e.g., statamozumab / MYO-29, e.g., having HCVR / LCVR amino acid sequences SEQ ID NO:626 and 627); anti-GDF8 antibodies as described in U.S. Patent 8,415,459 (e.g., RK22 / PF-0625616, e.g., having HCVR / LCVR amino acid sequences SEQ ID NO:628 and 629); anti-GDF8 antibodies as described in U.S. Patent 7,731,961 (e.g., JA-16, e.g., CDR having HCVR amino acid sequence SEQ ID NO:630); anti-GDF8 antibodies as described in U.S. Patent Nos. 8,496,934 or 7,888,486 (e.g., RK35, e.g., having HCVR / LCVR amino acid sequence SEQ ID NO:628 and 629); Anti-GDF8 antibodies (e.g., OGD1.0.0, e.g., having HCVR / LCVR amino acid sequences SEQ ID NO: 631 and 632), as described in U.S. Patent No. 8,992,913 (e.g., OGD1.0.0, e.g., having HCVR / LCVR amino acid sequences SEQ ID NO: 633 and 634); anti-GDF8 Fab molecules as described in European Patent 1773 041B1 and anti-GDF8 antibodies (e.g., C12, e.g., having HCVR / LCVR amino acid sequences SEQ ID NO: 635 and 636, C12-N93H and / or 510C2, having HCVR / LCVR amino acid sequences SEQ ID NO: 637 and 638) as described in U.S. Patent No. 7,632,499 (e.g., 41C1E4 / landogo group monoclonal antibody / LY2495655, e.g., having SEQ ID NO: 631 and 632); anti-GDF8 antibodies (e.g., 41C1E4 / landogo group monoclonal antibody / LY2495655, e.g., having SEQ ID NO: 633 and 634) as described in U.S. Patent No. 8,992,913 (e.g., having SEQ ID NO: 633 and 634); anti-GDF8 antibodies (e.g., 41C1E4 / landogo group monoclonal antibody / LY2495655, e.g., having SEQ ID NO: 633 and 634); anti-GDF8 antibodies (e.g., having SEQ ID NO: 633 and 634); anti-GDF8 antibodies (e.g., having SEQ ID NO: 6 (HCVR / LCVR amino acid sequences of NO: 639 and 640).In some embodiments, the anti-GDF8 antibody may have the full-length heavy chain and full-length light chain amino acid sequences of the landoogombo antibody, for example, SEQ ID Nos. 641 and 642. In some embodiments, the anti-GDF8 antibody may comprise the three heavy chain CDRs (HCDRs) and three light chain CDRs (LCDRs) of the landoogombo antibody, for example, as defined by Chothia according to SEQ ID Nos: 643 / 644 / 645 / 646 / 647 / 648, respectively.

[0125] In one embodiment, the anti-GDF8 antibody or its antigen-binding fragment comprises a heavy chain variable region (HCVR) having an amino acid sequence selected from the following: SEQ ID NO:2,18,34,50,66,82,98,114,130,146,162,178,194,210,226,242,258,274,290,306,360 and 376 or substantially similar sequences having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0126] In another embodiment, the anti-GDF8 antibody or its antigen-binding fragment comprises a light chain variable region (LCVR) amino acid sequence selected from SEQ ID NO:10,26,42,58,74,90,106,122,138,154,170,186,202,218,234,250,266,282,298,314,322,368, and384, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0127] In another embodiment, the anti-GDF8 antibody or its antigen-binding fragment comprises an HCVR amino acid sequence and an LCVR amino acid sequence, wherein the HCVR / LCVR sequence pair is selected from SEQ ID NO: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, 290 / 298, 306 / 314, 114 / 322, 360 / 368, and 376 / 384.

[0128] In another embodiment, the anti-GDF8 antibody or its antigen-binding fragment comprises an HCVR amino acid sequence and an LCVR amino acid sequence, wherein the HCVR / LCVR sequence pair is selected from (HCVR / LCVR): 21-E5 (SEQ ID NO: 34 / 42); 21-B9 (SEQ ID NO: 18 / 26); 21-E9 (SEQ ID NO: 98 / 106); 21-A2 (SEQ ID NO: 2 / 10); 22-D3 (SEQ ID NO: 50 / 58); 22-E6 (SEQ ID NO: 66 / 74); 22-G10 (SEQ ID NO: 82 / 90); 1A2 (SEQ ID NO: 226 / 234); 20B12 (SEQ ID NO: 274 / 282); 58C8 (SEQ ID NO: 242 / 250); 19F2 (SEQ ID NO: 258 / 266); 8D12-1 ... NO:114 / 122); 4E3-7 (SEQ ID NO:194 / 202); 9B11-12 (SEQ ID NO:162 / 170); 4B9 (SEQ ID NO:226 / 234); 1H4-5 (SEQ ID NO:210 / 218); 9B4-3 (SEQ ID NO:210 / 218); NO:178 / 186); 3E2-1 (SEQ ID NO:290 / 298); 4G3-25 (SEQ ID NO:306 / 314); 4B6-6 (SEQ ID NO:130 / 138); H4H1657N2 (SEQ ID NO:360 / 368); H4H1669P (SEQ ID NO:360 / 368); NO:376 / 384).

[0129] In another embodiment, the anti-GDF8 antibody or its antigen-binding fragment comprises a heavy chain complementarity-determining region 3 (HCDR3) domain and a light chain CDR3 (LCDR3) domain, wherein the HCDR3 domain has an amino acid sequence selected from SEQ ID NO:8,24,40,56,72,88,104,120,136,152,168,184,200,216,232,248,264,280,296,312,366, and382 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and the LCDR3 domain has an amino acid sequence selected from SEQ ID NO:8,24,40,56,72,88,104,120,136,152,168,184,200,216,232,248,264,280,296,312,366, and 382, ​​... The amino acid sequences of NO:16,32,48,64,80,96,112,128,144,160,176,192,208,224,240,256,272,288,304,320,328,374 and 390 or substantially similar sequences having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. In another embodiment, the anti-GDF8 antibody or its antigen-binding fragment comprises the HCDR3 / LCDR3 amino acid sequence pair selected from SEQ ID NO: 8 / 16, 24 / 32, 40 / 48, 56 / 64, 72 / 80, 88 / 96, 104 / 112, 120 / 128, 136 / 144, 152 / 160, 168 / 176, 184 / 192, 200 / 208, 216 / 224, 232 / 240, 248 / 256, 264 / 272, 280 / 288, 296 / 304, 312 / 320, 120 / 328, 366 / 374, and 382 / 390.

[0130] In another embodiment, the anti-GDF8 antibody or its antigen-binding fragment comprises heavy chain CDR1 (HCDR1) and CDR2 (HCDR2) domains and light chain CDR1 (LCDR1) and CDR2 (LCDR2) domains, wherein the HCDR1 domain has an amino acid sequence selected from SEQ ID NO:4,20,36,52,68,84,100,116,132,148,164,180,196,212,228,244,260,276,292,308,362, and378 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; the HCDR2 domain has an amino acid sequence selected from SEQ ID NO:4,20,36,52,68,84,100,116,132,148,164,180,196,212,228,244,260,276,292,308,362, and 378; The amino acid sequences NO:6,22,38,54,70,86,102,118,134,150,166,182,198,214,230,246,262,278,294,310,364, and 380, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; the LCDR1 domain has a sequence selected from SEQ ID NO. The amino acid sequences NO:12,28,44,60,76,92,108,124,140,156,172,188,204,220,236,252,268,284,300,316,324,370, and 386, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and the LCDR2 domain having a sequence selected from SEQ ID NO:12,28,44,60,76,92,108,124,140,156,172,188,204,220,236,252,268,284,300,316,324,370, and 386, or sequences substantially similar to these ... The amino acid sequences of NO:14,30,46,62,78,94,110,126,142,158,174,190,206,222,238,254,270,286,302,318,326,372 and 388 or substantially similar sequences having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. In another embodiment, the HCDR1, HCDR2, and HCDR3 domains have corresponding amino acid sequence combinations selected from SEQ ID NO: 36 / 38 / 40, 116 / 118 / 120, 228 / 230 / 232, 362 / 364 / 366, and 378 / 380 / 382; and the LCDR1, LCDR2, and LCDR3 domains have corresponding amino acid sequence combinations selected from SEQ ID NO: 44 / 46 / 48, 124 / 126 / 128, 236 / 238 / 240, 370 / 372 / 374, and 386 / 388 / 390.

[0131] In yet another embodiment, the heavy and light chain CDR domains of the anti-GDF8 antibody or its antigen-binding fragment (HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3) have amino acid sequence combinations selected from SEQ ID NO: 36 / 38 / 40 / 44 / 46 / 48 (e.g., 21-E5), 116 / 118 / 120 / 124 / 126 / 128 (e.g., 8D12), 228 / 230 / 232 / 236 / 238 / 240 (e.g., 1A2), 362 / 364 / 366 / 370 / 372 / 374 (e.g., H4H1657N2), and 378 / 380 / 382 / 386 / 388 / 390 (e.g., H4H1669P).

[0132] In another embodiment, the anti-GDF8 antibody or its antigen-binding fragment comprises heavy and light chain CDR domains within a heavy and light chain variable region (HCVR / LCVR) amino acid sequence pair, said sequence pair being selected from SEQ ID NO: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, 290 / 298, 306 / 314, 114 / 322, 360 / 368, and 376 / 384.

[0133] Methods and techniques for identifying CDRs in HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs in the specified HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia methods. See, for example, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273: 927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86: 9268-9272 (1989). Public databases can also be used to identify CDR sequences in antibodies.

[0134] In one embodiment, the anti-GDF8 antibody or its antigen-binding fragment comprises HCVR having an amino acid sequence encoded by a nucleic acid sequence selected from SEQ ID NO:1,17,33,49,65,81,97,113,129,145,161,177,193,209,225,241,257,273,289,305,359 and 375 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0135] In another embodiment, the anti-GDF8 antibody or its antigen-binding fragment comprises an LCVR having an amino acid sequence encoded by a nucleic acid sequence selected from SEQ ID NO: 9,25,41,57,73,89,105,121,137,153,169,185,201,217,233,249,265,281,297,313,321,367 and 383 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0136] In another embodiment, the anti-GDF8 antibody or its antigen-binding fragment comprises an HCVR / LCVR pair having an amino acid sequence encoded by a nucleic acid molecule pair selected from SEQ ID NO: 1 / 9, 17 / 25, 33 / 41, 49 / 57, 65 / 73, 81 / 89, 97 / 105, 113 / 121, 129 / 137, 145 / 153, 161 / 169, 177 / 185, 193 / 201, 209 / 217, 225 / 233, 241 / 249, 257 / 265, 273 / 281, 289 / 297, 305 / 313, 113 / 321, 359 / 367, and 375 / 383.

[0137] In another embodiment, the anti-GDF8 antibody or its antigen-binding fragment comprises an HCDR3 domain having an amino acid sequence encoded by a nucleic acid sequence selected from SEQ ID NO:7,23,39,55,71,87,103,119,135,151,167,183,199,215,231,247,263,279,295,311,365, and381, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an LCDR3 domain having an amino acid sequence encoded by a nucleic acid sequence selected from SEQ ID NO:7,23,39,55,71,87,103,119,135,151,167,183,199,215,231,247,263,279,295,311,365, and ... The amino acid sequence encoded by the nucleic acid sequence IDNO:15,31,47,63,79,95,111,127,143,159,175,191,207,223,239,255,271,287,303,319,327,373 and 389 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. In one embodiment, the HCDR3 / LCDR3 domain pair has an amino acid sequence encoded by a nucleic acid sequence pair selected from SEQ ID NO: 7 / 15, 23 / 31, 39 / 47, 55 / 63, 71 / 79, 87 / 95, 103 / 111, 119 / 127, 135 / 143, 151 / 159, 167 / 175, 183 / 191, 199 / 207, 215 / 223, 231 / 239, 247 / 255, 263 / 271, 279 / 287, 295 / 303, 311 / 319, 119 / 327, 365 / 373, and 381 / 389.

[0138] In another embodiment, the anti-GDF8 antibody or its antigen-binding fragment comprises HCDR1 and HCDR2 domains, and LCDR1 and LCDR2 domains, wherein the HCDR1 domain has an amino acid sequence encoded by a nucleic acid sequence selected from SEQ ID NO:3,19,35,51,67,83,99,115,131,147,163,179,195,211,227,243,259,275,291,307,361, and 377, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and the HCDR2 ... The amino acid sequence encoded by the nucleic acid sequence NO:5,21,37,53,69,85,101,117,133,149,165,181,197,213,229,245,261,277,293,309,363, and 379 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, wherein the LCDR1 domain comprises a sequence selected from SEQ ID NO:5,21,37,53,69,85,101,117,133,149,165,181,197,213,229,245,261,277,293,309,363, and 379. The amino acid sequence encoded by the nucleic acid sequence NO:11,27,43,59,75,91,107,123,139,155,171,187,203,219,235,251,267,283,299,315,323,369 and 385 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity, and the LCDR2 domain having an amino acid sequence encoded by a nucleic acid sequence selected from SEQ ID NO:13,29,45,61,77,93,109,125,141,157,173,189,205,221,237,253,269,285,301,317,325,371 and 387.

[0139] In another embodiment, the anti-GDF8 antibody or its antigen-binding fragment comprises heavy and light chain CDR domains (HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3) having an amino acid sequence combination encoded by the nucleic acid sequence set of SEQ ID NO: 35 / 37 / 39 / 43 / 45 / 47, 115 / 117 / 119 / 123 / 125 / 127, 227 / 229 / 231 / 235 / 237 / 239, 361 / 363 / 365 / 369 / 371 / 373 or 377 / 379 / 381 / 385 / 387 / 389.

[0140] In a preferred embodiment, the anti-GDF8 antibody or antigen-binding fragment that specifically binds to GDF8 comprises an HCDR containing the heavy chain variable region (HCVR) of SEQ ID NO:360 and an LCDR containing the light chain variable region (LCVR) of SEQ ID NO:368. In another embodiment, the anti-GDF8 antibody or antigen-binding fragment that specifically binds to GDF8 comprises three HCDRs and three LCDRs, wherein the HCDRs comprise SEQ ID NO:362, SEQ ID NO:364, and SEQ ID NO:366, and the LCDRs comprise SEQ ID NO:370, SEQ ID NO:372, and SEQ ID NO:374.

[0141] In another embodiment, the anti-GDF8 antibody or its antigen-binding fragment is a fully human or humanized antibody or antibody fragment that binds to GDF8 with an affinity of about 1 nM or less (expressed as a dissociation constant "KD"), as determined by surface plasmon resonance (e.g., BIACORE). TM The assay was performed. In some embodiments, the antibodies of the present invention exhibit the following K... D Approximately 700 pM or less; approximately 500 pM or less; approximately 320 pM or less; approximately 160 pM or less; approximately 100 pM or less; approximately 50 pM or less; approximately 10 pM or less; or approximately 5 pM or less.

[0142] In another embodiment, the anti-GDF8 antibody or its antigen-binding fragment is a fully human or humanized monoclonal antibody (mAb) that specifically binds to and inhibits human GDF8, and exhibits the following IC50 values. 50 Less than or equal to about 10 nM; about 5 nM or less; about 3 nM or less; about 2 nM or less; about 1 nM or less; about 500 pM or less; or about 200 pM or less, as determined by the GDF8-inducible luciferase assay.

[0143] In one embodiment, the anti-GDF8 antibody or its antigen-binding fragment has a modified glycosylation pattern. In some applications, modification to remove unwanted glycosylation sites, or antibodies lacking the fucose moiety present on the oligosaccharide chain, may be useful, for example, to enhance antibody-dependent cytotoxicity (ADCC) function (Shield et al. (2002) JBC277:26733). In other applications, galactosylation can be performed to modify complement-dependent cytotoxicity (CDC).

[0144] In another embodiment, the anti-GDF8 antibody or its antigen-binding fragment competes with another antibody comprising a combination of HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 domains for specific binding to GDF8, said domain combination having an amino acid sequence selected from SEQ ID NO: 36 / 38 / 40 / 44 / 46 / 48, 116 / 118 / 120 / 124 / 126 / 128, 228 / 230 / 232 / 236 / 238 / 240, 362 / 364 / 366 / 370 / 372 / 374 or 378 / 380 / 382 / 386 / 388 / 390. In another embodiment, the anti-GDF8 antibody or its antigen-binding fragment competes with another antibody containing the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, 290 / 298, 306 / 314, 114 / 322, 360 / 368, or 376 / 384 for specific binding to GDF8.

[0145] In another embodiment, the anti-GDF8 antibody or its antigen-binding fragment recognizes an epitope on GDF8 that is recognized by another antibody, said other antibody comprising a combination of HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 domains having an amino acid sequence selected from SEQ ID NO:36 / 38 / 40 / 44 / 46 / 48, 116 / 118 / 120 / 124 / 126 / 128, 228 / 230 / 232 / 236 / 238 / 240, 362 / 364 / 366 / 370 / 372 / 374 or 378 / 380 / 382 / 386 / 388 / 390. In another embodiment, the anti-GDF8 antibody or its antigen-binding fragment recognizes an epitope on GDF8 that is recognized by another antibody, said other antibody comprising the HCVR / LCVR amino acid sequence pairs of SEQ ID NO: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, 290 / 298, 306 / 314, 114 / 322, 360 / 368, or 376 / 384.

[0146] Activin A inhibitors

[0147] The present invention includes methods for altering body composition in an individual, inducing a reduction in fat mass and / or an increase in lean body mass, and methods for treating in an individual a disease or disorder characterized by an increase in fat mass, said methods including administering to the individual a composition containing an effective amount of an activin A inhibitor.

[0148] As used herein, "activin A inhibitor" refers to any active agent that binds to or interacts with human activin A and interferes with or inhibits the normal biological function of activin A in vitro or in vivo. Non-limiting examples of the activin A inhibitor class include small molecule activin A antagonists, nucleic acid-based inhibitors of activin A expression or activity (e.g., siRNA or antisense), peptide-based molecules that specifically interact with activin A (e.g., peptide antibodies), receptor molecules that specifically interact with activin A, activin A binding scaffold molecules, proteins comprising a ligand-binding portion of a receptor that specifically binds to activin A, and anti-activin A aptamers or portions thereof. In a preferred embodiment, the activin A inhibitor used in the context of this invention is an anti-activin A antibody or its antigen-binding fragment that specifically binds to human activin A. Inhibition caused by anti-activin A neutralizing and / or blocking antibodies need not be complete, as long as it is detectable using appropriate assays.

[0149] Activins are homodimeric and heterodimeric molecules containing βA and / or βB subunits. The βA subunit has the amino acid sequence of SEQ ID NO:617, and the βB subunit has the amino acid sequence of SEQ ID NO:619. Activin A is a homodimer of two βA subunits; activin B is a homodimer of two βB subunits; activin AB is a heterodimer of one βA subunit and one βB subunit. Anti-activin A antibodies or their antigen-binding fragments specifically bind to the βA subunits. Because the βA subunit is present in both activin A and activin AB molecules, "anti-activin A antibodies or their antigen-binding fragments" can specifically bind to both activin A and activin AB (through their interaction with the βA subunits). Therefore, anti-activin A antibodies or their antigen-binding fragments specifically bind to activin A, or bind to activin A and activin AB, but do not bind to other ActRIIB ligands, such as activin B, GDF3, GDF8, BMP2, BMP4, BMP7, BMP9, BMP10, GDF11, Nodal, etc.

[0150] In some embodiments, as described in U.S. Patent No. 9,718,881, an anti-activin A antibody or its antigen-binding fragment is used. Exemplary anti-activin A antibodies that can be used in the compositions and methods of the present invention include, for example, fully human anti-activin antibody H4H10446P2, also known as REGN2477 (e.g., an anti-activin A antibody comprising a heavy chain variable region and a light chain variable region having SEQ ID NO:162 and SEQ ID NO:146, respectively, as described in U.S. Patent No. 9,718,881).

[0151] Table 2 lists the heavy and light chain variable region amino acid sequence pairs of the selected anti-activin A antibodies and their corresponding antibody identifiers that can be used in the compositions and methods of the present invention. The corresponding nucleic acid sequence identifiers are listed in Table 3.

[0152] Table 2: Amino acid sequence identifiers of anti-activin A

[0153]

[0154]

[0155] Table 3: Anti-activin A nucleic acid sequence identifiers

[0156]

[0157] In one embodiment, the anti-activin A antibody or its antigen-binding fragment comprises a heavy chain variable region (HCVR) having an amino acid sequence selected from the following: SEQ ID NO: 393, 409, 425, 441, 457, 473, 489, 497, 505, 513, 521, 529, 545, 553, 561, 569, 577, 585 and 593 or substantially similar sequences having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0158] In another embodiment, the anti-activin A antibody or its antigen-binding fragment comprises a light chain variable region (LCVR) having an amino acid sequence selected from the following: SEQ ID NO:401,417,433,449,465,481,537 and 601 or substantially similar sequences having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0159] In another embodiment, the anti-activin A antibody or its antigen-binding fragment comprises an HCVR and LCVR (HCVR / LCVR) amino acid sequence pair selected from SEQ ID NO: 393 / 401, 409 / 417, 425 / 433, 441 / 449, 457 / 465, 473 / 481, 489 / 481, 497 / 481, 505 / 481, 513 / 481, 521 / 481, 529 / 537, 545 / 537, 553 / 537, 561 / 537, 569 / 537, 577 / 537, 585 / 537, and 593 / 601.

[0160] In another embodiment, the anti-activin A antibody or its antigen-binding fragment comprises a heavy chain CDR3 (HCDR3) domain having an amino acid sequence selected from the following: SEQ ID NO:399,415,431,447,463,479,495,503,511,519,527,535,551,559,567,575,583,591 and 599 or substantially similar sequences having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a light chain CDR3 (LCDR3) domain having an amino acid sequence selected from the following: SEQ ID NO:407,423,439,455,471,487,543 and 607 or substantially similar sequences having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0161] In another embodiment, the anti-activin A antibody or its antigen-binding fragment comprises the HCDR3 / LCDR3 amino acid sequence pair selected from SEQ ID NO: 399 / 407, 415 / 423, 431 / 439, 447 / 455, 463 / 471, 479 / 487, 495 / 487, 503 / 487, 511 / 487, 519 / 487, 527 / 487, 535 / 543, 551 / 543, 559 / 543, 567 / 543, 575 / 543, 583 / 543, 591 / 543, and 599 / 607.

[0162] In another embodiment, the anti-activin A antibody or its antigen-binding fragment comprises a heavy chain CDR1 (HCDR1) domain having an amino acid sequence selected from the following: SEQ ID NO:395,411,427,443,459,475,491,499,507,515,523,531,547,555,563,571,579,587 and 595 or substantially similar sequences having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a heavy chain CDR2 (HCDR2) domain having an amino acid sequence selected from the following: SEQ ID NO:397,413,429,445,461,477,493,501,509,517,525,533,549,557,565,573,581,589 and 597 or substantially similar sequences having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a light chain CDR1 (LCDR1) domain having an amino acid sequence selected from: SEQ ID NO:403,419,435,451,467,483,539 and 603 or substantially similar sequences having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a light chain CDR2 (LCDR2) domain having an amino acid sequence selected from: SEQ ID NO:405,421,437,453,469,485,541 and 605 or substantially similar sequences having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0163] In another embodiment, the anti-activin A antibody or its antigen-binding fragment respectively comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains having an amino acid sequence selected from the following: SEQ ID NO:395-397-8-403-405-407;411-413-415-419-421-423;36-429-431-435-437-439;443-445-447-451-453-455;459-461-463-467-469-4 71;475-477-479-483-485-487;491-493-495-483-485-487;499-501 -503-483-485-487;507-509-511-483-485-487;515-517-519-483-48 5-487; 523-525-527-483-485-487; 531-533-535-539-541-543; 547-549-551-539-541-543; 555-557-559-539-541-543(H4H10446P2); 563-565-567-539-541-543; 571-573-575-539-541-543; 579-581-583-539-541-543; 587-589-591-539-541-543; and 595-597-599-603-605-607.

[0164] In another embodiment, the anti-activin A antibody or its antigen-binding fragment comprises heavy and light chain CDR domains contained in heavy and light chain variable region (HCVR / LCVR) sequence pairs selected from SEQ ID NO:393 / 401,409 / 417,425 / 433,441 / 449,457 / 465,473 / 481,489 / 481,497 / 481,505 / 481,513 / 481,521 / 481,529 / 537,545 / 537,553 / 537,561 / 537,569 / 537,577 / 537,585 / 537 and 593 / 601.

[0165] In one embodiment, the anti-activin A antibody or its antigen-binding fragment comprises HCVR having an amino acid sequence encoded by a nucleic acid sequence selected from SEQ ID NO:392,408,424,440,456,472,488,496,504,512,520,528,544,552,560,568,576,584 and 592 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0166] In another embodiment, the anti-activin A antibody or its antigen-binding fragment comprises an LCVR having an amino acid sequence encoded by a nucleic acid sequence selected from SEQ ID NO:400,416,432,448,464,480,536 and 600 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0167] In another embodiment, the anti-activin A antibody or its antigen-binding fragment comprises HCVR and LCVR, wherein the HCVR / LCVR pair has an amino acid sequence encoded by a nucleic acid sequence pair selected from SEQ ID NO: 392 / 400, 408 / 416, 424 / 432, 440 / 448, 456 / 464, 472 / 480, 488 / 480, 496 / 480, 504 / 480, 512 / 480, 520 / 480, 528 / 536, 544 / 536, 552 / 536, 560 / 536, 568 / 536, 576 / 536, 584 / 536, and 592 / 600.

[0168] In another embodiment, the anti-activin A antibody or its antigen-binding fragment comprises an HCDR3 domain having an amino acid sequence encoded by a nucleic acid sequence selected from SEQ ID NO:398,414,430,446,462,478,498,502,510,518,526,534,550,558,566,574,582,590, and 598, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an LCDR3 domain having an amino acid sequence encoded by a nucleic acid sequence selected from SEQ ID NO:406,422,435,454,470,486,542, and 606, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In one embodiment, the HCDR3 / LCDR3 domain group has an amino acid sequence encoded by a nucleic acid sequence pair selected from SEQ ID NO: 398 / 406, 414 / 422, 430 / 438, 446 / 454, 462 / 470, 478 / 486, 494 / 486, 502 / 486, 510 / 486, 518 / 486, 526 / 486, 534 / 542, 550 / 542, 558 / 542, 566 / 542, 574 / 542, 582 / 542, 590 / 542, and 598 / 606.

[0169] In another embodiment, the anti-activin A antibody or its antigen-binding fragment comprises HCDR1 and HCDR2 domains and LCDR1 and LCDR2 domains, wherein the HCDR1 domain has an amino acid sequence encoded by a nucleic acid sequence selected from SEQ ID NO:394,410,426,442,458,474,490,498,506,514,522,530,546,554,562,570,578,586, and594 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and the HCDR2 domain has an amino acid sequence encoded by a nucleic acid sequence selected from SEQ ID NO:394,410,426,442,458,474,490,498,506,514,522,530,546,554,562,570,578,586, and 594, ... The LCDR1 domain comprises an amino acid sequence encoded by a nucleic acid sequence selected from SEQ ID NO:396,412,428,444,460,476,492,500,508,516,524,532,548,556,564,572,580,588, and 596, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and the LCDR2 domain comprises an amino acid sequence encoded by a nucleic acid sequence selected from SEQ ID NO:402,418,434,450,466,482,538, and 602, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and the LCDR2 domain comprises an amino acid sequence encoded by a nucleic acid sequence selected from SEQ ID NO:402,418,434,450,466,482,538, and 602, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. The amino acid sequences encoded by the nucleic acid sequences NO:404,420,436,452,468,484,540 and 604 or sequences substantially similar to them having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0170] In another embodiment, the anti-activin A antibody or its antigen-binding fragment comprises heavy and light chain CDR (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3) domains having an amino acid sequence encoded by a nucleic acid sequence selected from: SEQ ID NO:394 / 396 / 398 / 402 / 404 / 406,410 / 412 / 414 / 418 / 420 / 422,426 / 428 / 430 / 434 / 436 / 438,442 / 444 / 446 / 450 / 452 / 454,458 / 460 / 462 / 466 / 468 / 470,474 / 476 / 478 / 482 / 484 / 486,490 / 492 / 494 / 482 / 484 / 486,498 / 500 / 502 / 482 / 484 / 486,506 / 508 / 510 / 482 / 484 / 486,514 / 516 / 51 8 / 482 / 484 / 486,522 / 524 / 526 / 482 / 484 / 486,530 / 532 / 534 / 538 / 540 / 542,546 / 548 / 550 / 538 / 540 / 542,554 / 556 / 558 / 538 / 540 / 542,562 / 564 / 566 / 538 / 540 / 542,570 / 572 / 574 / 538 / 540 / 542,578 / 580 / 582 / 538 / 540 / 542,586 / 588 / 590 / 538 / 540 / 542 and 594 / 596 / 598 / 602 / 604 / 606.

[0171] In one embodiment, the anti-activin A antibody or its antigen-binding fragment comprises the HCVR and LCVR (HCVR / LCVR) amino sequence pair of SEQ ID NO:553 / 537, and the anti-GDF8 antibody or its antigen-binding fragment comprises the HCVR and LCVR (HCVR / LCVR) amino sequence pair of SEQ ID NO:360 / 368.

[0172] In another embodiment, the anti-activin A antibody or its antigen-binding fragment respectively comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains having the following amino acid sequence: SEQ ID NO: 555-557-559-539-541-543 (H4H10446P2), and the anti-GDF8 antibody or its antigen-binding fragment respectively comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains having the following amino acid sequence: SEQ ID NO: 362 / 364 / 366 / 370 / 372 / 374 (e.g., H4H1657N2).

[0173] In another embodiment, the anti-activin A antibody or its antigen-binding fragment has a modified glycosylation pattern. In some applications, modification to remove unwanted glycosylation sites, or antibodies lacking the fucose moiety present on the oligosaccharide chain, may be useful, for example, for enhancing antibody-dependent cytotoxicity (ADCC) function (Shield et al. (2002) JBC277:26733). In other applications, galactosylation can be performed to modify complement-dependent cytotoxicity (CDC).

[0174] Compared to the corresponding germline sequences, the fully human anti-activin A and / or anti-GDF8 antibodies described herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework regions and / or CDR regions of the heavy and light chain variable domains. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available from, for example, public antibody sequence databases. In another embodiment, the compositions and methods of the present invention use antibodies and their antigen-binding fragments derived from any amino acid sequence disclosed herein, wherein one or more amino acids in one or more framework regions and / or CDR regions are reverted to the corresponding germline residues or conserved amino acid substitutions (natural or non-natural) of the corresponding germline residues (such sequence changes are referred to herein as "germline reversion mutations"). Those skilled in the art can readily generate numerous antibody and antigen-binding fragments containing one or more individual germline reversion mutations or combinations thereof, starting from the heavy and light chain variable region sequences described herein. In some embodiments, all framework and / or CDR residues within the VH and / or VL domains are mutated back to the germline sequence. In other embodiments, only certain residues are mutated back to the germline sequence, for example, only mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. Furthermore, antibody-antigen binding fragments used in the compositions and methods of the present invention may contain any combination of two or more germline reversion mutations in the framework region and / or CDR region, i.e., certain individual residues mutate back to the germline sequence while retaining certain other residues different from the germline sequence. Once obtained, antibody-antigen binding fragments containing one or more germline reversion mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as applicable), reduced immunogenicity, etc. Antibody-antigen binding fragments obtained in this general manner are included within the scope of the present invention.

[0175] In other embodiments, the compositions and methods of the present invention use anti-GDF8 antibodies and / or anti-activin A antibodies (or antigen-binding fragments thereof) comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences described herein, said variants having one or more conserved substitutions. For example, in some embodiments, the anti-GDF8 antibodies and / or anti-activin A antibodies used in the compositions and methods of the present invention have HCVR, LCVR, and / or CDR amino acid sequences having, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conserved amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences described herein.

[0176] Bispecific antibodies

[0177] Bispecific antibodies (bsAbs) combine the specificity of two antibodies and simultaneously bind to different antigens or epitopes. Two or more antigen recognition elements are engineered into a single antibody. In one embodiment of the method of the present invention, the composition comprises an antibody containing a GDF8-specific binding domain and an activin A-specific binding domain. As used herein, the term "specific binding domain" includes peptides or components thereof that are: (i) antigen-binding fragments of an antibody molecule, (ii) peptides (e.g., peptide antibodies) that specifically interact with a particular antigen, and / or (iii) ligand-binding moieties of receptors that specifically bind to a particular antigen. For example, a bispecific antibody may be included, with one arm containing a first heavy chain variable region / light chain variable region (HCVR / LCVR) pair that specifically binds to GDF8, and the other arm containing a second HCVR / LCVR pair that specifically binds to activin A.

[0178] Bispecific antibodies can be prepared according to known methods, including chemical crosslinking, hybridoma / cell hybridoma, mortis, CrossMab, dual variable domain immunoglobulin, recombinant engineering (tandem single-chain variable fragment / biantibody), and docking and locking. Other exemplary bispecific forms that can be used in this invention include, but are not limited to, IgG-scFv fusions, dual variable domain (DVD)-Ig, common light chains, CrossFab, (SEED)body, leucine zippers, Duobody, IgG1 / IgG2, dual-action Fab (DAF)-IgG, and Mab2 bispecific forms (see, for example, Klein et al., mAbs 4:6,1-11 (2012) and references cited therein for a review of the foregoing forms). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugations, for example, where non-natural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates, which then self-assemble into multimeric complexes having defined composition, titer, and geometry. (See, for example, Kazane et al., J AmChem Soc. 135(1):340-346(2013)).

[0179] Specific binding

[0180] The term "specific binding" as used in this article refers to the formation of a complex between an antigen-specific binding protein or antigen-specific binding domain and a specific antigen, characterized by a dissociation constant (K). D The concentration is 500 pM or lower, and it does not bind to other unrelated antigens under normal testing conditions. "Unrelated antigens" are proteins, peptides, or polypeptides that share less than 95% amino acid identity with each other. Methods for determining whether two molecules specifically bind to each other are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. For example, antigen-specific binding proteins or antigen-specific binding domains as used in the context of this invention include K at concentrations below about 500 pM, below about 400 pM, below about 300 pM, below about 200 pM, below about 100 pM, below about 90 pM, below about 80 pM, below about 70 pM, below about 60 pM, below about 50 pM, below about 40 pM, below about 30 pM, below about 20 pM, below about 10 pM, below about 5 pM, below about 4 pM, below about 2 pM, below about 1 pM, below about 0.5 pM, below about 0.2 pM, below about 0.1 pM, or below about 0.05 pM. D Antibodies or antigen-binding fragments that bind to specific antigens (e.g., GDF8, activin A) or portions thereof, as measured by surface plasmon resonance assays.

[0181] Antibody binding (to antigen) can be based on K, which represents the affinity strength. D To quantify. K DThe lower the value, the higher the antibody binding affinity. The term "K" is used in this article. D "This refers to the equilibrium dissociation constant of a specific antibody-antigen interaction. Surface plasmon resonance can be used to determine ligand binding, such as antibody-antigen interactions."

[0182] The term "surface plasmon resonance" as used in this article refers to an optical phenomenon that allows for the analysis of real-time, biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using BIACORE. TM Systems (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0183] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specifically binding to immunoglobulins or T-cell receptors. In some embodiments, the epitope determinant comprises chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments, may have specific three-dimensional structural features and / or specific charge features. An epitope is a region where an antigen is bound by an antibody. In some embodiments, when an antibody preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules, it is said to specifically bind to the antigen. For example, when K... D Less than or equal to 10 -8 M, less than or equal to 10 -9 M or less than or equal to 10 -10 When M occurs, the antibody is considered to specifically bind to the antigen.

[0184] Preparation of human antibodies

[0185] Methods for producing monoclonal antibodies, including fully human monoclonal antibodies, are known in the art. Any of these known methods can be used in the context of this invention to prepare human antibodies that specifically bind to GDF8 and / or activin A.

[0186] Use VELOCIMMUNE TM The technique, or any other known method for producing monoclonal antibodies, initially isolates high-affinity chimeric antibodies against GDF8 and / or activin A, which have a human variable region and a mouse constant region. As described in the Experimental Section below, the antibody is characterized and desired features, including affinity, selectivity, epitopes, etc., are selected. The mouse constant region is replaced with the desired human constant region to produce the fully human antibody of the present invention, such as wild-type or modified IgG1 or IgG4. Although the selected constant region may vary depending on the specific application, high-affinity antigen binding and target-specific features are present in the variable region.

[0187] Typically, the antibodies used in the methods of this invention have very high affinity when measured by binding to antigens immobilized on a solid phase or in a solution phase, typically having about 10. -12 approximately 10 -9 M of K D .

[0188] Pharmaceutical Compositions and Administration

[0189] This invention includes a method for altering an individual's body composition. As used herein, the phrase "altering body composition" refers to a change in one or more of lean body mass, fat mass, and / or bone mass in an individual. In some embodiments, an individual's body composition can be altered by administering an effective amount of a GDF8 inhibitor and an activator A inhibitor to the individual. Lean body mass can be, for example, thigh muscle volume, limb lean body mass, or total lean body mass. In some aspects, thigh muscle volume can refer to the volume of thigh muscle tissue excluding intramuscular adipose tissue and large blood vessels. In some aspects, thigh muscle volume can refer to the volume of thigh muscle tissue including intramuscular adipose tissue and large blood vessels. In some aspects, limb lean body mass can be calculated using, for example, the aLBM equation. In some aspects, limb lean body mass can be calculated by summing the lean body mass of the arms and legs. Fat mass can be, for example, total fat mass, male-type fat mass, the sum of intramuscular and perimuscular adipose tissue (IMAT), subcutaneous adipose tissue volume, the sum of fat mass in the arms and legs, and intramuscular adipose tissue in the thigh. Bone mass can be, for example, total bone mineral density (BMD) or total bone mineral content (BMC). In some embodiments, altering body composition includes increasing muscle mass and / or decreasing fat mass. In some embodiments, altering body composition includes simultaneously increasing muscle mass and decreasing fat mass. In some embodiments, altering body composition includes simultaneously increasing muscle mass and decreasing fat mass without decreasing bone mass. In some embodiments, altering body composition includes increasing bone mineral content. In some embodiments, altering body composition includes decreasing total fat mass, masculinity fat mass, and / or subcutaneous fat mass. In some embodiments, altering body composition includes decreasing total fat mass, masculinity fat mass, and / or subcutaneous fat mass without decreasing the volume of intramuscular fat tissue in the thigh.

[0190] This invention includes methods for altering body composition, such as inducing a reduction in an individual's body fat mass, and methods for treating diseases or disorders characterized by increased body fat mass, said methods comprising administering to an individual a first composition containing an effective amount of a GDF8 inhibitor and a second composition containing an effective amount of an activin A inhibitor. The first and second compositions may be administered to the individual simultaneously or sequentially. The first and second compositions may also be combined into a third composition prior to administration. Therefore, in some embodiments, a composition containing both a GDF8 inhibitor and an activin A inhibitor may be administered to the individual. The GDF8 inhibitor in such a composition may be, for example, an anti-GDF8 antibody. The activin A inhibitor in such a composition may be, for example, an anti-activin A antibody.

[0191] Many suitable formulations can be found in all pharmaceutical chemists' known formularies: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, and lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN). TM DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, polyethylene glycol emulsions (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycol. Any of the foregoing mixtures may be used in the treatments and therapies according to the invention, provided that the active ingredient in the formulation is not inactivated by the formulation via the route of administration, and that the formulation is physiologically compatible and tolerable. See also Powell et al., “Compendium of excipients for parenteral formulations”, PDA (1998), JPharm Sci Technol 52: 238-311.

[0192] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262: 4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered via any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucosal linings (e.g., oral mucosa, rectal and intestinal mucosa), and can also be administered together with other biologically active agents.

[0193] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using standard needles and syringes. Additionally, regarding subcutaneous delivery, pen delivery devices are readily applicable for delivering the pharmaceutical compositions of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices typically utilize replaceable cartridges containing the pharmaceutical composition. After all the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there are no replaceable cartridges. Instead, the disposable pen delivery device is pre-filled with the pharmaceutical composition, which is held in a reservoir within the device. After the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0194] Many reusable pen-type and auto-injector delivery devices have applications in the subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, AUTOPEN. TM (Owen Mumford, Inc., Woodstock, UK), DISSETRONIC TM pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25 TM pen, HUMALOG TM pen, HUMALIN 70 / 30 TM pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (NovoNordisk, Copenhagen, Denmark), BD TM pen(Becton Dickinson,Franklin Lakes,NJ)、OPTIPEN TM OPTIPEN PRO TM OPTIPEN STARLET TM and OPTICLIK TM(Sanofi-Aventis, Frankfurt, Germany), to name just a few. Examples of disposable pen-type delivery devices with application in subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, SOLOSTAR. TM pen (Sanofi-Aventis), FLEXPEN TM (Novo Nordisk) and KWIKPEN TM (Eli Lilly), SURECLICK TM Autoinjector (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP) and HUMIRA TM Pen (Abbott Labs, AbbottPark IL), to name just a few.

[0195] In some cases, the pharmaceutical compositions of the present invention can be delivered in a controlled-release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials may be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled-release system may be placed near the target of the composition, thus requiring only a portion of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, above, Vol. 2, pp. 115-138). Other controlled-release systems are discussed in the review in Langer, 1990, Science 249:1527-1533.

[0196] Injectable products can include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, infusion, etc. These injectable products can be prepared by known methods. For example, injectable products can be prepared by dissolving, suspending, or emulsifying, for example, antibodies or salts thereof described above in a sterile aqueous or oily medium commonly used for injection. As an aqueous medium for injection, there are, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, which can be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (a polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. As an oily medium, there are, for example, sesame oil, soybean oil, which can be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, etc. The injection prepared in this way is preferably filled in a suitable ampoule.

[0197] Advantageously, the pharmaceutical compositions described above for oral or parenteral use are prepared into unit-dose dosage forms suitable for containing a specific dose of the active ingredient. Such unit-dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0198] dose

[0199] The amount of active ingredient (e.g., anti-GDF8 antibody and / or anti-activin A antibody) that can be administered to an individual is typically a therapeutically effective amount. The term "effective amount" is the concentration or amount of an active ingredient, such as an antibody or an antigen-binding fragment of an antibody, that results in achieving the specific stated purpose. The terms "effective amount" and "therapeutically effective amount" are used interchangeably and refer to the concentration or amount of an active ingredient, such as an antibody or an antigen-binding fragment thereof, that effectively achieves the stated therapeutic effect. The (therapeutic) effective amount can be determined empirically.

[0200] As used herein, the phrase "therapeutic effective dose" or "effective dose" refers to the dose of antigen-specific binding proteins and / or antigen-binding molecules (e.g., antibodies) that result in a detectable reduction in fat mass. In some embodiments, the effective dose may also result in an increase in one or more of the following parameters: body weight, muscle mass (e.g., tibialis anterior [TA] muscle mass, gastrocnemius [GA] muscle mass, quadriceps [Quad] muscle mass, limb lean body mass, etc.), muscle volume (e.g., thigh muscle volume), muscle strength / power and / or muscle function, and glucose tolerance.

[0201] The "therapeutic effective amount" or "effective amount" of a GDF8 inhibitor (e.g., anti-GDF8 antibody) and / or activin A inhibitor (e.g., anti-activin A antibody) includes, for example, an amount of GDF8 inhibitor and / or activin A inhibitor that, when administered to an individual, results in a reduction of total fat mass of at least about 2% to 8%, at least 2.5% to 6%, at least 3% to 4%, or at least about 2.0%, at least about 2.5%, at least about 3.0%, or at least about 3.5% or more. For example, the "therapeutic effective amount" or "effective amount" of a GDF8 inhibitor (e.g., anti-GDF8 antibody) and / or activin A inhibitor (e.g., anti-activin A antibody) includes, for example, an amount of GDF8 inhibitor and / or activin A inhibitor that, when administered to an individual, results in a reduction of total fat mass of at least about 3.5% or more.

[0202] In some embodiments, a "therapeuticly effective amount" or "effective amount" of a GDF8 inhibitor (e.g., anti-GDF8 antibody) and / or an activator A inhibitor (e.g., anti-activator A antibody) includes, for example, an amount of GDF8 inhibitor and / or activator A inhibitor that, when administered to an individual, results in a reduction of male-type body fat mass of at least about 2% to 8%, at least 2.5% to 6%, at least 3% to 4%, or at least about 2.0%, at least about 2.5%, at least about 3.0%, or at least about 3.5% or more. For example, a "therapeuticly effective amount" or "effective amount" of a GDF8 inhibitor (e.g., anti-GDF8 antibody) and / or an activator A inhibitor (e.g., anti-activator A antibody) includes, for example, an amount of GDF8 inhibitor and / or activator A inhibitor that, when administered to an individual, results in a reduction of male-type body fat mass of at least about 3.5%.

[0203] In some implementations, the amount also resulted in an increase in TA or GA muscle mass of at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% or more compared to individuals receiving control treatment.

[0204] In some implementations, the "therapeutic effective amount" or "effective amount" of a GDF8 inhibitor (e.g., anti-GDF8 antibody) and / or activin A inhibitor (e.g., anti-activin A antibody) includes, for example, an amount of GDF8 inhibitor and / or activin A inhibitor that, when administered to an individual, results in an increase in thigh muscle volume of at least about 2% to 8%, 2.5% to 6%, 3% to 4%, or at least 2.0%, at least 2.5%, at least 3.0%, or at least 3.5% or more.

[0205] In some implementations, the "therapeutic effective amount" or "effective amount" of a GDF8 inhibitor (e.g., anti-GDF8 antibody) and / or activin A inhibitor (e.g., anti-activin A antibody) includes, for example, an amount of GDF8 inhibitor and / or activin A inhibitor that, when administered to an individual, results in an increase in total lean body mass of at least about 2% to 8%, 2.5% to 6%, 3% to 4%, or at least 2.0%, at least 2.5%, at least 3.0%, or at least 3.5% or more.

[0206] In some implementations, the "therapeutic effective amount" or "effective amount" of a GDF8 inhibitor (e.g., anti-GDF8 antibody) and / or activin A inhibitor (e.g., anti-activin A antibody) includes, for example, an amount of GDF8 inhibitor and / or activin A inhibitor that, when administered to an individual, results in an increase in limb lean body mass of at least about 2% to 8%, 2.5% to 6%, 3% to 4%, or at least 2.0%, at least 2.5%, at least 3.0%, or at least 3.5% or more.

[0207] In some implementations, the amount also resulted in an increase in TA or GA muscle mass of at least 2%, 3%, 5%, 10%, 15%, 20%, 25% or more compared to individuals receiving control treatment.

[0208] In some implementations, the therapeutically effective amount of anti-GDF8 antibody, anti-activin A antibody, or bispecific antibody that specifically binds to GDF8 and activin A can be from about 0.05 mg to about 600 mg; for example, about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, etc. The corresponding antibodies are approximately 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, 500 mg, 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, or 600 mg. Dosage can vary depending on the individual's age and size, the target disease, symptoms, and route of administration. The frequency and duration of treatment can be adjusted according to the severity of the condition.

[0209] The amount of antibody (e.g., anti-GDF8 antibody, anti-activin A antibody, or bispecific antibody that specifically binds to GDF8 and activin A) contained in each dose can be expressed as the number of milligrams of antibody per kilogram of patient body weight (i.e., mg / kg). For example, the anti-GDF8 antibody, anti-activin A antibody, and / or anti-GDF8 / anti-activin A bispecific antibody in the first, second, or third composition applied according to the method of the present invention can be administered to the patient at a dose of about 0.0001 to about 50 mg / kg of patient body weight (e.g., 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, 5.0 mg / kg, 5.5 mg / kg, 6.0 mg / kg, 6.5 mg / kg, 7.0 mg / kg, 7.5 mg / kg, 8.0 mg / kg, 8.5 mg / kg, 9.0 mg / kg, 9.5 mg / kg, 10.0 mg / kg, 10.5 mg / kg, 11.0 mg / kg, 11.5 mg / kg, etc.).

[0210] In some embodiments, an effective amount of a GDF8 inhibitor (e.g., an anti-GDF8 antibody) may include a dosing regimen selected from at least 0.1 mg / kg to about 10 gm / kg, 1 mg / kg to about 1 gm / kg, and 10 mg / kg to 100 mg / kg. In some embodiments, an effective amount of an activin A inhibitor (e.g., an anti-activin A antibody) may include a dosing regimen selected from at least 0.1 mg / kg to about 10 gm / kg, 1 mg / kg to about 1 gm / kg, and 10 mg / kg to 100 mg / kg.

[0211] In another embodiment, an effective amount of a GDF8 inhibitor (e.g., anti-GDF8 antibody) may include a single-dose administration regimen selected from about 0.01 to about 20 mg / kg body weight, about 0.1 to about 10 mg / kg body weight, and about 0.1 to about 5 mg / kg body weight. In another embodiment, an effective amount of an activin A inhibitor (e.g., anti-activin A antibody) may include a single-dose administration regimen selected from about 0.01 to about 20 mg / kg body weight, about 0.1 to about 10 mg / kg body weight, and about 0.1 to about 5 mg / kg body weight. In one specific aspect, an effective amount of anti-GDF8 antibody is about 2 mg / kg to 10 mg / kg, 4 mg / kg to 8 mg / kg, or about 6 mg / kg body weight, and an effective amount of anti-activin A antibody is 0.5 mg / kg to 15 mg / kg, 2 mg / kg to 12 mg / kg, about 3 mg / kg, or about 10 mg / kg body weight.

[0212] In some embodiments, the first, second, and third compositions applied according to the method of the present invention may contain equal amounts of a GDF8 inhibitor (e.g., anti-GDF8 antibody) and / or an activin A inhibitor (e.g., anti-activin A antibody). Alternatively, the amount of GDF8 inhibitor (e.g., anti-GDF8 antibody) in the composition may be less than or greater than the amount of activin A inhibitor (e.g., anti-activin A antibody). The effective amount of GDF8 inhibitor (e.g., anti-GDF8 antibody) when combined with an activin A inhibitor (e.g., anti-activin A antibody) may be lower than the effective amount in the composition alone. When combined with a GDF8 inhibitor (e.g., anti-GDF8 antibody), the effective amount of activin A inhibitor (e.g., anti-activin A antibody) may be lower than the effective amount in the composition alone. Those skilled in the art will be able to determine the appropriate amounts of each component in the composition required to produce the desired therapeutic effect using conventional experiments.

[0213] All aspects of this disclosure

[0214] This disclosure provides compositions, kits, and methods for reducing body fat mass (inducing fat reduction) in an individual using a GDF8 inhibitor and an activator A inhibitor. This disclosure also provides compositions, kits, and methods for treating diseases, disorders, and / or conditions in an individual that are associated with or characterized by increased body fat mass using a GDF8 inhibitor and an activator A inhibitor. In a preferred embodiment, the GDF8 inhibitor is an antibody that specifically binds to GDF8 or an antigen-binding fragment thereof.

[0215] Treatment

[0216] This invention includes methods for altering body composition by specifically binding GDF8 and / or activin A, such as methods for inducing a decrease in body fat mass in an individual, methods for increasing muscle mass in an individual, and methods for treating diseases or disorders characterized by increased body fat mass. For example, this invention includes methods for inducing a decrease in body fat mass in an individual, methods for inducing an increase in muscle mass in an individual, and methods for treating diseases or disorders characterized by increased body fat mass in an individual, said methods being performed by administering to the individual i) a composition comprising an anti-GDF8 antibody and an anti-activin A antibody, or ii) a composition comprising both an anti-GDF8 antibody and an anti-activin A antibody, or iii) a composition comprising a bispecific antibody, said bispecific antibody comprising a first variable domain and a second variable domain, the first variable domain comprising an HCVR / LCVR pair specifically binding to GDF8, and the second variable domain comprising an HCVR / LCVR pair specifically binding to activin A. Any of the GDF8 inhibitors and / or activin A inhibitors disclosed or involved herein may be used in these aspects of the invention.

[0217] In methods including administering a GDF8 inhibitor and an activator A inhibitor to an individual, the GDF8 inhibitor (e.g., an anti-GDF8 antibody) and the activator A inhibitor (e.g., an anti-activator A antibody) may be administered to the individual simultaneously or substantially simultaneously, for example, in a single therapeutic dose (third composition) or in two separate doses (first and second compositions), administered simultaneously or at intervals of less than about 5 minutes. Alternatively, the GDF8 inhibitor and the activator A inhibitor (first and second compositions) may be administered to the individual sequentially, for example, in separate therapeutic doses at intervals of more than about 5 minutes.

[0218] The reduction in fat mass of an individual according to the method of the present invention can be a reduction in total fat mass as measured by DXA (dual-energy X-ray absorptiometry).

[0219] In another embodiment, the reduction in fat mass in an individual according to the method of the invention is a reduction in male-type fat mass (i.e., visceral fat associated with the upper / midbody), as measured by DXA (dual-energy X-ray absorptiometry). In male-type obesity, an individual stores fat around his or her abdominal region. Male-type obesity can also occur in other areas of the upper body, such as the upper chest (front or back), neck region, and even the shoulders. Individuals with male-type obesity have a greater risk of obesity-related diseases / disorders such as heart disease and metabolic syndrome. The likelihood of developing gout, arterial-related diseases (due to hypertension), and various cancers is also associated with a central type of fat distribution in individuals exhibiting male-type obesity.

[0220] Body fat assessments vary in precision and accuracy. Common anthropometric measurements include weight, waist circumference, and skinfold measurements using calipers. More sophisticated methods include bioelectrical impedance analysis (BIA), BOD POD, and dual-energy X-ray absorptiometry (DEXA or DXA). DXA is particularly accurate and effective because it considers bone mineral content when estimating body fat and muscle. DEXA scans can assess different areas of fat distribution to determine the male / female body fat ratio, which differs from body mass index. DXA can measure total fat mass, total muscle mass, visceral fat (fat surrounding organs), intramuscular fat (fat between muscles), total bone mineral density, and can even provide regional breakdowns. Finally, DXA can accurately assess body fat distribution associated with increased insulin resistance.

[0221] Avoid side effects

[0222] This invention includes methods for altering body composition, such as inducing a reduction in an individual's body fat mass, and methods for treating diseases or disorders characterized by increased body fat mass, said methods comprising administering a GDF8 inhibitor and an activin A inhibitor to an individual without causing adverse side effects associated with the administration of a molecule binding multiple (e.g., three or more) ActRIIB ligands, as described in, for example, U.S. Patent No. 8,871,209. For example, a clinical molecule called ACE-031 (Acceleon Pharma, Inc., Cambridge, MA) is a multimer composed of the extracellular portion of ActRIIB fused to an IgG Fc domain (this molecule is also referred to herein as "ActRIIB-Fc"). ActRIIB-Fc binds to GDF8 and other ActRIIB ligands, such as activin A, activin B, GDF11, BMP9, BMP10, and TGFβ, and is known to cause various adverse side effects when administered to human patients. For example, in a phase Ib escalation dose study, administration of ACE-031 to postmenopausal women showed an undesirable increase in hemoglobin and a decrease in FSH levels. Furthermore, the Phase II study of ACE-031 in pediatric patients with muscular dystrophy was discontinued due to adverse effects including nasal and gingival bleeding. Vascular dilation was also observed in patients treated with ActRIIB-Fc. The effects of ACE-031 on boys with Duchenne muscular dystrophy (DMD) confirmed a trend toward increased lean body mass and decreased fat mass, but non-muscle-related adverse events led to the decision to discontinue the study (Campbell et al. 2017 Muscle Nerve 55:458-464). Specific inhibition of GDF8 and activin A (e.g., by administration of anti-GDF8 and anti-activin A antibodies), without inhibition of other ActRIIB ligands such as activin B, GDF11, BMP9, BMP10, and TGFβ, results in an increased reduction in fat mass without causing the adverse side effects associated with non-specific activin-binding agents such as ActRIIB-Fc.

[0223] Application plan

[0224] According to some embodiments of the invention, multiple doses of the compositions of the invention (e.g., compositions comprising a GDF8 inhibitor and / or an activator A inhibitor (e.g., an anti-GDF8 antibody and / or an anti-activator A antibody, or a bispecific antibody against GDF8 and activator A)) can be administered to an individual over a defined time period. A method according to one aspect of the invention comprises sequentially administering multiple doses of the compositions of the invention to an individual. As used herein, "sequentially administering" means administering each dose of the compositions of the invention to an individual at different time points, such as on different days separated by predetermined intervals (e.g., hours, days, weeks, or months). The invention includes a method comprising sequentially administering to a patient an initial dose of a first and / or a second composition; or a third composition; followed by one or more second doses of the first and / or second compositions; or a third composition; and optionally followed by one or more third doses of the first and / or second compositions; or a third composition.

[0225] The terms "initial dose," "second dose," and "third dose" refer to the order in which the compositions of the present invention are administered. Thus, the "initial dose" is the dose administered at the start of a treatment regimen (also known as the "baseline dose"); the "second dose" is the dose administered after the initial dose; and the "third dose" is the dose administered after the second dose. The initial, second, and third doses may all contain the same amount of the active ingredient, such as anti-GDF8 antibody and / or anti-activin A antibody, but typically differ from each other in terms of frequency of administration. However, in some embodiments, the amount of active ingredient contained in the initial, second, and / or third doses will differ from each other during treatment (e.g., adjusted upwards or downwards as appropriate).

[0226] In one exemplary embodiment of the invention, each second and / or third dose is administered 1 to 30 days after the immediately preceding dose (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more). As used herein, the phrase "immediately preceding dose" refers to the dose of the composition of the invention administered to an individual in a sequence of administrations, prior to the next dose in the administration sequence, without intermediate doses.

[0227] A method according to this aspect of the invention may include administering any number of second and / or third doses of the composition of the invention to a patient. For example, in some embodiments, only a single second dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) second doses are administered to the patient. Similarly, in some embodiments, only a single third dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) third doses are administered to the patient.

[0228] In embodiments involving multiple second doses, each second dose may be administered at the same frequency as the other second doses. For example, each second dose may be administered to the patient 1 to 29 days immediately following the preceding dose. Similarly, in embodiments involving multiple third doses, each third dose may be administered at the same frequency as the other third doses. For example, each third dose may be administered to the patient 1 to 60 days immediately following the preceding dose. Alternatively, the frequency of administration of the second and / or third doses to the patient may vary throughout the treatment regimen. The frequency of administration may also be adjusted by the physician during treatment, depending on the individual patient's needs following clinical examination.

[0229] In one embodiment, an individual may undergo an initial DXA (Direct Detection Analysis), followed by a DXA follow-up with a composition comprising anti-GDF8 antibody and anti-activin A antibody (or a composition comprising anti-GDF8 antibody and a composition comprising anti-activin A antibody). If the fat content in subsequent DXA is not significantly reduced (compared to the initial DXA), the individual may receive the composition again. In another embodiment, the subsequent dosage and frequency of administration may be varied based on the results of subsequent DXA.

[0230] Combination therapy

[0231] According to some embodiments, the method of the present invention includes administering to an individual one or more other therapeutic agents, which may advantageously be combined with a composition containing a GDF8 inhibitor and / or an activator A inhibitor. The term "combination" as used herein means the administration of the other therapeutic agent before, after, or simultaneously with the pharmaceutical composition containing a GDF8 inhibitor and / or an activator A inhibitor. The term "combination" also includes the sequential or simultaneous administration of a GDF8 inhibitor, an activator A inhibitor, or both of these and a second therapeutic agent. The term "therapeutic agent" also means including a specific therapy.

[0232] Other therapeutic agents may include, for example, additional GDF8 antagonists / inhibitors, additional activin A antagonists / inhibitors, growth factor inhibitors, immunosuppressants, metabolic inhibitors, enzyme inhibitors, and cytotoxic agents / cell inhibitors, IL-1 antagonists (including, for example, the IL-1 antagonists described in US 6,927,044), IL-6 antagonists, IL-6R antagonists (including, for example, the anti-IL-6R antibody described in US 7,582,298), IL-13 antagonists, tumor necrosis factor (TNF) antagonists, IL-8 antagonists, IL-9 antagonists, and I... L-17 antagonists, IL-5 antagonists, IgE antagonists, CD48 antagonists, IL-31 antagonists (including, for example, those described in US7,531,637), thymic stromal lymphopoietin (TSLP) antagonists (including, for example, those described in US2011 / 027468), interferon-γ (IFNγ) antibiotics, topical corticosteroids, tacrolimus, pimecrolimus, cyclosporine, azathioprine, methotrexate, sodium cromoglycate, proteolytic enzyme inhibitors, systemic corticosteroids, systemic immunotherapy, antihistamines, chemotherapy, phototherapy, or combinations thereof.

[0233] In a further embodiment, the invention is characterized in that the additional therapeutic agent is selected from (1) 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors, such as cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, etc.; (2) inhibitors of cholesterol uptake and / or bile acid reabsorption; (3) niacin, which increases lipoprotein catabolism; and (4) fibrates or amphiphilic carboxylic acids, which lower low-density lipoprotein (LDL). (5) Activators of LXR transcription factors that play a role in cholesterol elimination, such as 22-hydroxycholesterol, or fixed combinations such as ezetimibe and simvastatin; fixed combinations of statins with bile resins (e.g., cholestyramine, colestipol, colesvelam), niacin and statin compounds (e.g., niacin and lovastatin); or fixed combinations with other lipid-lowering agents such as omega-3 fatty acid ethyl esters (e.g., omacor).

[0234] In a further embodiment, the second therapeutic agent is selected from one or more other inhibitors / antagonists of glucagon or glucagon receptors, and inhibitors of other molecules, such as inhibitors of ANGPTL8 (e.g., anti-ANGPTL8 antibody), and inhibitors of other molecules, such as ANGPTL3 (e.g., anti-ANGPTL3 antibody), ANGPTL4, ANGPTL5, ANGPTL6, apolipoprotein C-III (also known as APOC3; see, for example, inhibitors of APOC3 described in US8530439, US7750141, US7598227, and volanesorsen, also known as ISIS-APOCIIIRx), and proprotein convertase subtilisin / kexin type 9 (PCSK9), which are involved in lipid metabolism, particularly cholesterol and / or triglyceride homeostasis. Inhibitors of these molecules include small molecules, antisense molecules, and antibodies that specifically bind to these molecules and block their activity.

[0235] In a further implementation, if necessary, additional therapeutic agents may be selected from analgesics, anti-inflammatory agents, including nonsteroidal anti-inflammatory drugs (NSAIDs), such as Cox-2 inhibitors, to improve and / or alleviate symptoms of the underlying condition.

[0236] Additional therapeutic agents may be administered before, simultaneously with, or after the first and / or second composition; or the third composition described herein. For the purposes of this disclosure, the administration regimen may be considered as administration of the anti-GDF8 antibody and / or anti-activin A antibody in combination with the second therapeutically active component. Example

[0237] The following examples are provided to provide those skilled in the art with a complete disclosure and description of how to prepare and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure the accuracy of the numerical values ​​used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0238] Example 1. In clinical studies, anti-GDF8 alone increased total lean body mass by up to 3%: Phase 2 data for sarcopenia.

[0239] A randomized, double-blind, placebo-controlled phase 2 clinical trial of interventional therapy was conducted in patients aged 70 years and older (including men with sarcopenia and postmenopausal women). Patients were treated with subcutaneous anti-GDF8 antibody REG1033 (H4H1657N2) alone for 12 weeks, either 100 mg anti-GDF8 antibody, Q4-week subcutaneous (sc) (n=62); 300 mg anti-GDF8 antibody, Q4-week subcutaneous (sc) (n=64); or 300 mg anti-GDF8 antibody, Q2-week subcutaneous (sc) (n=59), or placebo (n=65). Figure 1 As shown, when REGN1033 was used alone at each of the three doses, patients showed a significant increase in total lean body mass after 12 weeks compared to placebo, as indicated by... Figure 1 As shown in Table 4.

[0240] Table 4. Changes in total lean body mass percentage from week 12 with anti-GDF8 REGN1033 alone.

[0241]

[0242] The effects on strength and function differ. Anti-GDF8 is generally safe and well-tolerated (responses, if any, are mild). Table 4 shows that patients receiving 100 mg or 300 mg doses of REGN1033 exhibited a significant increase in total lean body mass, as a percentage change from placebo to week 12. In this study, the anti-GDF8 antibody REGN1033 alone increased total lean body mass by up to 3%. The 300 mg regimen also resulted in a reduction in total fat mass and androgenetic adipose mass.

[0243] Example 2. First dose-escalation study in humans of the combination of anti-GDF8 and anti-activin.

[0244] A randomized, double-blind, placebo-controlled, dose-escalation study was initiated to evaluate the tolerability and effects of the combination of intravenous anti-GDF8 antibody and anti-activin A antibody relative to the individual components on body composition.

[0245] The primary objective of this study was to evaluate the safety and tolerability of anti-activin A antibodies (e.g., H4H10446P2 = REGN2477) alone and in combination with anti-GDF8 antibodies (e.g., H4H1657N2 = REGN1033) in healthy postmenopausal women aged 45 to 70 years.

[0246] Secondary objectives of the study included: assessing the effects of REGN2477 alone, REGN1033 alone, and the combination of REGN2477 and REGN1033 on thigh muscle volume as determined by magnetic resonance imaging (MRI), and assessing the effects of REGN2477 alone, REGN1033 alone, and the combination of REGN2477 and REGN1033 on total and regional body composition as determined by dual-energy X-ray absorptiometry (DXA).

[0247] Research Design

[0248] This study was a randomized, double-blind, placebo-controlled, dose-escalation study to evaluate the safety, tolerability, and pharmacodynamics of intravenous REGN2477 (anti-activin A) alone and in combination with REGN1033 (anti-GDF8) in healthy postmenopausal women.

[0249] A total of 48 subjects were randomly assigned to one of the following four consecutively increasing REGN2477 IV dose groups. Eight subjects were randomly assigned to each of the first three groups (groups A, B, and C) in a 6:2 ratio, and 24 subjects were randomly assigned to group D in a 1:1:1:1 ratio (6 subjects in each group).

[0250] Group A: 4 subjects receiving REGN1033 (6 mg / kg IV) + low-dose REGN2477 (1 mg / kg IV) or 2 subjects receiving placebo.

[0251] Group B: 4 subjects receiving REGN1033 (6 mg / kg IV) + REGN2477 (medium dose, 3 mg / kg IV) or 2 subjects receiving placebo.

[0252] Group C: 4 subjects receiving REGN1033 (6 mg / kg IV) + high-dose REGN2477 (10 mg / kg IV) or 2 subjects receiving placebo.

[0253] Group D: REGN1033 (6 mg / kg IV) + REGN2477 (high dose, 10 mg / kg IV) placebo, REGN2477 (10 mg / kg IV) or REGN1033 (6 mg / kg IV)

[0254] Subjects received a single intravenous dose of one or both of the following: anti-GDF8 antibody REGN1033 and / or anti-activin A antibody REGN2477. In the preliminary analysis, the placebo and high-dose combination groups were combined in each group, resulting in 12 subjects receiving placebo and 12 subjects receiving the high-dose combination, as shown below. Figure 2A As shown. Figure 2AThe dosing regimen shown is for subsequent Figures 2B to 17 Each of the studies shown in the table.

[0255] Subjects participated in a 28-day screening period, followed by baseline and treatment follow-up on day 1, with a follow-up period of 113 days.

[0256] Efficacy and safety analysis

[0257] The full analysis set (FAS) includes all randomized subjects; it is based on assigned treatment (randomization). Efficacy endpoints are analyzed using the FAS. The safety analysis set (SAF) includes all randomized subjects receiving any investigational drug; it is based on the treatment received (e.g., the treatment administered). Treatment adherence / administration and all clinical safety variables are analyzed using the SAF.

[0258] The efficacy variables included: thigh muscle tissue volume, excluding and including intramuscular adipose tissue and large blood vessels, as determined by magnetic resonance imaging (MRI); total lean body mass as determined by dual X-ray absorptiometry (DXA); limb lean body mass as determined by DXA (calculated using the LBM equation); and total fat mass as determined by DXA.

[0259] Based on Table 5 below, the demographic and baseline characteristics of the subjects were balanced across the treatment groups:

[0260] Table 5. Balanced baseline characteristics among treatment groups

[0261]

[0262] Statistical methods

[0263] An analysis of covariance (ANCOVA) model was used, with the treatment group as a fixed effect and baseline values ​​as a continuous covariate, to analyze the percentage change and change in the efficacy variable from baseline to week 4 or 8 in the full analysis set (FAS). The least-squares means for each treatment group at weeks 4 and 8, with corresponding standard errors, confidence intervals, and p-values, were provided from the model for treatment comparisons. Placebo subjects were pooled into groups. Missing efficacy data were not entered. Adjustments for multiple tests were not applied in this study.

[0264] result

[0265] A total of 48 subjects were randomized to receive the study drug and complete the study. One subject in the high-dose REGN2477+REGN1033 group had an interrupted study drug infusion due to the adverse event of 'infusion site swelling'.

[0266] effect

[0267] In week 8, the blockade of both activator A and GDF8 in the combination increased thigh muscle volume and reduced fat mass, such as Figure 2B and 2C As shown. The greatest effect was observed at the highest dose combination.

[0268] Figure 2B A bar chart showing the percentage change in thigh muscle volume as measured by MRI at 8 weeks following a single dose in postmenopausal women is displayed. The numbers show the change compared to placebo. * indicates nominal p < 0.05 relative to placebo; **** indicates nominal p < 0.0001 relative to placebo. Single doses (mg / kg) of anti-activin A antibody and / or anti-GDF8 antibody are shown below the bars. Treatment with the combination of medium and high doses of REGN2477 + REGN1033 resulted in a significant dose-related increase in thigh muscle volume compared to placebo (p < 0.05; p < 0.0001, respectively). At week 8, subjects in the high-dose group showed a percentage change of up to 7.73% from baseline, compared to 0.88% with placebo.

[0269] Figure 2C The bar chart shows the percentage change in total fat mass as measured by DXA 8 weeks after a single dose in postmenopausal women. The figures show the change compared to placebo. * indicates a nominal p < 0.05 relative to placebo; **** indicates a nominal p < 0.0001 relative to placebo. Single doses (mg / kg) of anti-activin A antibody and / or anti-GDF8 antibody are shown below the bars. Treatment with the high-dose REGN2477 + REGN1033 combination resulted in a significant reduction in total fat mass compared to placebo (p < 0.05).

[0270] like Figure 3 As shown, the combination of anti-activin A antibody REGN2477 and anti-GDF8 antibody REGN1033 resulted in a dose-dependent increase in thigh muscle volume. Results of thigh muscle volume measured by MRI, excluding intermuscular adipose tissue and large blood vessels, are also summarized in Table 6 below. Compared with placebo, treatment with medium and high doses of REGN2477 + REGN1033 resulted in a significant increase in thigh muscle volume (p < 0.05), as shown in Table 6.

[0271] Table 6. Thigh muscle volume as measured by MRI*

[0272]

[0273] *Excludes intermuscular fat tissue and major blood vessels

[0274] As shown in Table 4, compared with placebo, treatment with the combination of medium and high doses of REGN2477+REGN1033 resulted in a significant dose-related increase in thigh muscle volume.

[0275] Figure 4 Increased thigh muscle volume was consistently observed in individual subjects treated with the combination of anti-activin A and anti-GDF8. Different lines within each treatment group represent different individuals.

[0276] The effect of the combination of anti-activin A antibody REGN2477 and anti-GDF8 antibody REGN1033 on limb lean body mass (the sum of lean body mass in the arms and legs) is similar to that of other antibodies. Figure 5 The results for thigh muscle volume shown are similar. The results for limb lean body mass as determined by DXA are also summarized in Table 7. The medium- and high-dose combinations significantly increased limb lean body mass compared to placebo, as shown in Table 7.

[0277] Table 7. Lean body mass of limbs, as measured by DXA

[0278]

[0279] like Figure 6 As shown, blocking activin A and GDF8 resulted in a reduction in total fat mass, as assessed by DXA. At week 8, by DXA measurement, the high-dose combination of anti-GDF8 antibody REGN1033 and anti-activin A antibody REGN2477 significantly reduced total fat mass compared to placebo (*p<0.05). Total fat mass results measured by DXA are also summarized in Table 8 below.

[0280] Table 8. Total Fat Mass Measured by DXA

[0281]

[0282] * p<0.05

[0283] As assessed by DXA, blockade of activin A and GDF8 was also found to be associated with a reduction in male-type body fat mass. Figure 7 As shown. At week 8, the high-dose combination of anti-GDF8 antibody REGN1033 and anti-activin A antibody REGN2477 significantly reduced male-type fat mass compared with placebo (*p<0.05), as measured by DXA.

[0284] Further efficacy results are shown in Figures 8 to 17 The results are summarized in the table below. High, medium, and low doses of REGN2477+REGN1033 are shown in the table below. Figure 2A middle.

[0285] Compared with placebo, the medium-dose (p<0.05) and high-dose (p<0.001) REGN2477+REGN1033 groups showed a significant increase in thigh muscle volume (excluding intramuscular adipose tissue and large blood vessels). Figure 8 At 8 weeks, thigh muscle volume increased by 7.73% in the high-dose REGN2477+REGN1033 group compared to 0.88% in the placebo group (nominal p<0.001). Moderate-dose REGN2477+REGN1033 and REGN1033 alone also significantly increased thigh muscle volume compared to placebo. A dose-responsive increase in thigh muscle volume was consistently observed in individual subjects treated with the combination therapy. (Data not shown).

[0286] Compared with placebo, the high-dose REGN2477+REGN1033 group showed a significant increase in total lean body mass (measured by DXA) (p<0.05). Figure 9 ).

[0287] Compared with placebo, the combined REGN2477+REGN1033 treatment group showed a significant increase in limb lean body mass (calculated by the aLBM equation) in each quadrant (low dose p<0.05, medium and high dose groups p<0.001). Figure 10 At week 8, compared with 0.76% in the placebo group, the limb lean body mass in the medium-dose REGN2477+REGN1033 group increased by 7.15% from baseline. Similarly, compared with 0.76% in the placebo group, the limb lean body mass in the high-dose REGN2477+REGN1033 group increased by 5.7% from baseline.

[0288] Total fat mass was significantly reduced in the high-dose REGN2477+REGN1033 treatment group; compared with placebo (0.5%), total fat mass was reduced by 3.92% (high-dose group) (nominal p<0.05). Figure 11 ).

[0289] At 4 and 8 weeks, compared with placebo, thigh muscle volume (including intramuscular adipose tissue and major blood vessels) was significantly increased in both the intermediate- and high-dose REGN2477+REGN1033 groups and the REGN1033 group. Figure 12 At 4 weeks, compared with placebo, low-dose REGN2477+REGN1033 also showed a significant increase in thigh muscle volume (including intramuscular adipose tissue and large blood vessels).

[0290] Compared with placebo, in each of the REGN2477+REGN1033 combination treatment groups, limb lean body mass (the sum of arms and legs) was significantly increased at both weeks 4 and 8 (p<0.05). Figure 13).

[0291] The male-type fat mass was also significantly reduced in the high-dose REGN2477+REGN1033 treatment group. Figure 14 Compared to no reduction in the placebo group, the high-dose REGN2477+REGN1033 group showed a 6.6% reduction in male-type body fat mass.

[0292] Compared with placebo, the intramuscular fat tissue volume (cm²) in the thigh muscle tissue group was significantly higher at 8 weeks in the high-dose REGN2477+REGN1033 group. 3 Significantly increased (p<0.05) Figure 15 ).

[0293] In the high-dose group, a reduction in adipose tissue was observed in the sum of intramuscular and peripheral vascular adipose tissue (IMAT). Figure 16 In the low and medium dose groups, a reduction in subcutaneous adipose tissue was observed. Figure 17 Conversely, the high-dose group showed an increase of 8% in intramuscular fat tissue in the thigh muscles, compared to a 4% decrease in the placebo group. Figure 17 ).

[0294] Compared with placebo, REGN2477+REGN1033 significantly increased all major measures of muscle volume and lean body mass in the high-dose group at both weeks 4 and 8; the effect at week 4 was generally less pronounced than that at week 8 (Table 9). The percentage change in key body composition measurements at week 8 is summarized in Table 9 below (full analysis set, LS mean and SE presentation).

[0295] Table 9. Summary of Body Composition Changes in Week 8

[0296]

[0297]

[0298] *p<0.05; **p<0.001

[0299] In Table 9 above, changes relative to baseline and differences from placebo are based on the least squares (LS) mean of an ANCOVA model, where baseline is a covariate and treatment is a fixed factor. Standard error (SE) and p-values ​​are also taken from ANCOVA. Nominal p-values ​​are recorded.

[0300] Bone mineral density (BMD) and bone mineral content (BMC) were determined by DXA, as shown in Table 10. At high doses, R2477+R1033 increased bone mineral content as determined by DXA, while total bone mineral density remained unchanged (Table 10).

[0301] Table 10 shows the sum of fat mass in the arms and legs. At high doses, R2477+R1033 reduced the sum of fat mass in the arms and legs (p<0.05).

[0302] Data from the primary endpoint analysis are shown in Table 10 below.

[0303] Table 10: Preliminary Analysis of Efficacy Endpoints

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315] Note: Least squares (LS) mean, standard error (SE), and p-values ​​are taken from ANCOVA. The model includes baseline measurements as covariates and treatment as a fixed factor.

[0316] *Indicates p-value < 0.05; **Indicates p-value < 0.001.

[0317] Compared with placebo, the R2477+R1033 combination significantly increased thigh muscle volume and total lean body mass in both the medium-dose and high-dose groups. Figure 8 and 9 ), significantly increased limb lean body mass in all dosage groups ( Figure 10Furthermore, the high-dose group showed a significant reduction in total fat mass and male-type fat mass (Table 10). At weeks 4 and 8, compared to placebo, the high-dose group of R2477+R1033 significantly increased all major measures of muscle volume and lean body mass; the effect at week 4 was generally less pronounced than at week 8 (Table 10). At week 8, the high-dose anti-activin A R2477+anti-GDF8R1033 group showed an increased percentage change in total bone mineral content, as determined by DXA, compared to placebo, while total bone mineral density remained unchanged (Table 10).

[0318] Security

[0319] Except for one serious treatment-associated adverse event (TEAE) of "radial fracture" reported by a placebo subject, all treatment-associated adverse events (TEAEs) were of mild to moderate severity. There were no serious adverse events, no deaths, and no interruptions due to TEAEs. Headache was the most common TEAE in each treatment group, occurring in 58.3% of all subjects and 50% of the placebo subjects. Muscle cramps, nausea, and oral ulcers were other common TEAEs in the REGN2477+REGN1033 group, occurring in 25% or more of subjects in the combined R2477+R1033 group; these TEAEs occurred less frequently in the placebo group, but no clear dose-response relationship was observed. There were no significant signs of bleeding or diarrhea, i.e., adverse events associated with activin receptor blockade. One epistaxis (preferred term for nosebleed) TEAE occurred in the R2477+R1033 dose group – resolved within 9 minutes.

[0320] A review of the potential clinical significance value (PCSV) revealed no significant differences between REG2477+REGN1033 and placebo in laboratory, vital signs, and ECG categories, which would indicate a negative impact of REGN2477+REGN1033. In the laboratory, vital signs, and ECG categories, 0–2 subjects in the combined REGN2477+REGN1033 dose group (N = 24) had PCSV; however, the percentage of subjects with PCSV was equal to or lower than that found in the placebo group. No post-treatment PCSV related to liver function tests was observed.

[0321] in conclusion

[0322] In healthy postmenopausal women, a single intravenous dose of REGN2477+REGN1033 increased thigh muscle volume, total lean body mass, and limb lean body mass. A surprising finding was the consistency of the thigh muscle changes: all individuals exposed to this combination showed an increase in thigh muscle volume, such as... Figure 4As shown. Furthermore, a single intravenous dose of REGN2477+REGN1033 reduced total fat, particularly in male-type patients. Treatment with REGN1033 alone increased thigh muscle volume.

[0323] Overall, in this clinical study, REGN2477, REGN1033, and REGN2477+REGN1033 were considered to have an acceptable safety profile and were well tolerated. No serious adverse events occurred.

[0324] The scope of this invention is not limited to the specific embodiments described herein. In fact, various modifications to the invention will become apparent to those skilled in the art from the foregoing description and drawings, in addition to those described herein. All such modifications will fall within the scope of the appended claims.

Claims

1. A method for altering body composition in an individual for non-therapeutic and non- prophylactic purposes, the method comprising administering to the individual a first composition comprising an effective amount of a GDF8 inhibitor, wherein the GDF8 inhibitor is an isolated antibody or antigen-binding fragment thereof that specifically binds GDF8, wherein the antibody or antigen-binding fragment that specifically binds GDF8 comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, respectively, having the amino acid sequences of SEQ ID NOs: 362 / 364 / 366, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, respectively, having the amino acid sequences of SEQ ID NOs: 370 / 372 / 374, and a second composition comprising an effective amount of an Activin A inhibitor, wherein the Activin A inhibitor is an isolated antibody or antigen-binding fragment thereof that specifically binds Activin A, wherein the antibody or antigen-binding fragment that specifically binds Activin A comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, respectively, having the amino acid sequences of SEQ ID NOs: 555 / 557 / 559, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, respectively, having the amino acid sequences of SEQ ID NOs: 539 / 541 / 543, wherein the individual is a healthy individual, and wherein the altering body composition comprises inducing a reduction in fat mass in the individual.

2. The method of claim 1, wherein the altering body composition further comprises inducing an increase in muscle mass in the individual.

3. A method for altering body composition in an individual for non-therapeutic and non- prophylactic purposes, the method comprising administering to the individual a composition comprising an effective amount of a GDF8 inhibitor and an effective amount of an Activin A inhibitor, wherein the GDF8 inhibitor is an isolated antibody or antigen-binding fragment thereof that specifically binds GDF8, wherein the antibody or antigen-binding fragment that specifically binds GDF8 comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, respectively, having the amino acid sequences of SEQ ID NOs: 362 / 364 / 366, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, respectively, having the amino acid sequences of SEQ ID NOs: 370 / 372 / 374, and wherein the Activin A inhibitor is an isolated antibody or antigen-binding fragment thereof that specifically binds Activin A, wherein the antibody or antigen-binding fragment that specifically binds Activin A comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, respectively, having the amino acid sequences of SEQ ID NOs: 555 / 557 / 559, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, respectively, having the amino acid sequences of SEQ ID NOs: 539 / 541 / 543, wherein the individual is a healthy individual, and wherein the altering body composition comprises inducing a reduction in fat mass in the individual.

4. The method of claim 3, wherein the altering body composition further comprises inducing an increase in muscle mass in the individual.

5. The method of any one of claims 1-4, wherein the effective amount of the GDF8 inhibitor comprises an administration regimen of 0.01 mg / kg to 10 g / kg.

6. The method of any one of claims 1-4, wherein the effective amount of the Activin A inhibitor comprises an administration regimen of 0.01 mg / kg to 10 g / kg.

7. The method of any one of claims 1-4, wherein the effective amount of the GDF8 inhibitor comprises an administration regimen of a single dose of 0.01 to 20 mg / kg body weight.

8. The method of any one of claims 1-4, wherein the effective amount of the Activin A inhibitor comprises an administration regimen of a single dose of 0.01 to 20 mg / kg body weight.

9. The method of any one of claims 1-4, wherein the effective amount of the GDF8 inhibitor is 6 mg / kg body weight of the individual.

10. The method of any one of claims 1-4, wherein the effective amount of the Activin A inhibitor is 3 mg / kg or 10 mg / kg body weight of the individual.

11. The method of claim 1 or 2, wherein the first composition is formulated as an intravenous, subcutaneous, or oral dosage form.

12. The method of claim 1 or 2, wherein the second composition is formulated as an intravenous, subcutaneous, or oral dosage form.

13. The method of claim 1 or 2, wherein the first and second compositions are combined into a third composition, and wherein the third composition is formulated as an intravenous, subcutaneous, or oral dosage form.

14. The method of any one of claims 3 or 4, wherein the composition is formulated as an intravenous, subcutaneous, or oral dosage form.

15. The method of any one of claims 1-4, further comprising determining the total fat mass and / or android fat mass of the individual prior to administration.

16. The method of claim 15, further comprising determining the total fat mass and / or android fat mass of the individual after administration, and administering the composition until the total fat mass and / or android fat mass of the individual is reduced by 2% to 8%.

17. The method of claim 15, further comprising determining the total fat mass and / or android fat mass of the individual after administration, and administering the composition until the total fat mass and / or android fat mass of the individual is reduced by at least 3.5%.

18. The method of any one of claims 1-4, wherein the antibody or antigen-binding fragment that specifically binds GDF8 comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 360 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

368.

19. The method of any one of claims 1-4, wherein the antibody or antigen-binding fragment that specifically binds Activin A comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 553 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

537.

20. The method of any one of claims 1-4, wherein the amount of activin A inhibitor is 100% to 400% of the amount of GDF8 inhibitor by weight.

21. The method of claim 20, wherein the amount of activin A inhibitor is 1.5 to 2.0 times the amount of the GDF8 inhibitor by weight.

22. The method of any one of claims 1-4, wherein the reduction in fat mass in the individual is a reduction in total fat mass as measured by DXA (dual-energy X-ray absorptiometry).

23. The method of any one of claims 1-4, wherein the reduction in fat mass in the individual is a reduction in android fat mass as measured by DXA (dual-energy X-ray absorptiometry).

24. The method of any one of claims 1-4, wherein the individual experiences an increase in muscle volume after administration.

25. The method of any one of claims 1-4, wherein the individual does not have a muscle wasting disorder or disease.

26. Use of a GDF8 inhibitor and an activin A inhibitor in the manufacture of a medicament for treating a disease or disorder characterized by increased fat mass, wherein the GDF8 inhibitor is an isolated antibody or antigen-binding fragment thereof that specifically binds GDF8, wherein the antibody or antigen-binding fragment that specifically binds GDF8 comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 362 / 364 / 366, respectively, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 370 / 372 / 374, respectively, and the activin A inhibitor is an isolated antibody or antigen-binding fragment thereof that specifically binds activin A, wherein the antibody or antigen-binding fragment that specifically binds activin A comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 555 / 557 / 559, respectively, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 539 / 541 / 543, respectively.

27. The use of claim 26, wherein the GDF8 inhibitor is an isolated antibody or antigen-binding fragment thereof that specifically binds GDF8, wherein the antibody or antigen-binding fragment that specifically binds GDF8 comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 362 / 364 / 366, respectively, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 370 / 372 / 374, respectively, and the activin A inhibitor is an isolated antibody or antigen-binding fragment thereof that specifically binds activin A, wherein the antibody or antigen-binding fragment that specifically binds activin A comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 555 / 557 / 559, respectively, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 539 / 541 / 543, respectively.

27. Use of a first composition comprising an effective amount of a GDF8 inhibitor, wherein the GDF8 inhibitor is an isolated antibody or antigen-binding fragment thereof that specifically binds GDF8, wherein the antibody or antigen-binding fragment that specifically binds GDF8 comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, respectively, having the amino acid sequences of SEQ ID NOs: 362 / 364 / 366, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, respectively, having the amino acid sequences of SEQ ID NOs: 370 / 372 / 374, and a second composition comprising an effective amount of an Activin A inhibitor, wherein the Activin A inhibitor is an isolated antibody or antigen-binding fragment thereof that specifically binds Activin A, wherein the antibody or antigen-binding fragment that specifically binds Activin A comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, respectively, having the amino acid sequences of SEQ ID NOs: 555 / 557 / 559, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, respectively, having the amino acid sequences of SEQ ID NOs: 539 / 541 / 543, in the manufacture of a product for altering body composition in an individual, wherein the individual is a healthy individual, and wherein the altering body composition comprises inducing a decrease in fat mass in the individual.

28. The use of claim 27, wherein the altering body composition comprises inducing an increase in muscle mass in the individual.

29. Use of a composition comprising an effective amount of a GDF8 inhibitor and an effective amount of an Activin A inhibitor, wherein the GDF8 inhibitor is an isolated antibody or antigen-binding fragment thereof that specifically binds GDF8, wherein the antibody or antigen-binding fragment that specifically binds GDF8 comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, respectively, having the amino acid sequences of SEQ ID NOs: 362 / 364 / 366, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, respectively, having the amino acid sequences of SEQ ID NOs: 370 / 372 / 374, and the Activin A inhibitor is an isolated antibody or antigen-binding fragment thereof that specifically binds Activin A, wherein the antibody or antigen-binding fragment that specifically binds Activin A comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, respectively, having the amino acid sequences of SEQ ID NOs: 555 / 557 / 559, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, respectively, having the amino acid sequences of SEQ ID NOs: 539 / 541 / 543, in the manufacture of a product for altering body composition in an individual, wherein the individual is a healthy individual, and wherein the altering body composition comprises inducing a decrease in fat mass in the individual.

30. The use of claim 29, wherein the altering body composition comprises inducing an increase in muscle mass in the individual.

31. The use of any one of claims 27-30, wherein the effective amount of the GDF8 inhibitor comprises an administration regimen of 0.01 mg / kg to 10 g / kg.

32. The use of any one of claims 27-30, wherein the effective amount of the Activin A inhibitor comprises an administration regimen selected from 0.01 mg / kg to 10 g / kg.

33. The use of any one of claims 27-30, wherein the effective amount of the GDF8 inhibitor comprises an administration regimen of a single dose of 0.01 to 20 mg / kg body weight.

34. The use of any one of claims 27-30, wherein the effective amount of the Activin A inhibitor comprises an administration regimen of a single dose of 0.01 to 20 mg / kg body weight.

35. The use of any one of claims 27-30, wherein the effective amount of the GDF8 inhibitor is 6 mg / kg body weight of the individual.

36. The use of any one of claims 27-30, wherein the effective amount of the Activin A inhibitor is 3 mg / kg or 10 mg / kg body weight of the individual.

37. The use of any one of claims 27 or 28, wherein the first composition is formulated as an intravenous, subcutaneous, or oral dosage form.

38. The use of any one of claims 27 or 28, wherein the second composition is formulated as an intravenous, subcutaneous, or oral dosage form.

39. The use of any one of claims 27 or 28, wherein the first and second compositions are combined into a third composition, wherein the third composition is formulated as an intravenous, subcutaneous, or oral dosage form.

40. The use of any one of claims 29 or 30, wherein the composition is formulated as an intravenous, subcutaneous, or oral dosage form.

41. The use of any one of claims 27-30, further comprising determining the total fat mass and / or android fat mass of the individual prior to administration.

42. The use of claim 41, further comprising determining the total fat mass and / or android fat mass of the individual after administration, and administering the composition until the total fat mass and / or android fat mass of the individual is reduced by 2% to 8%.

43. The use of claim 41, further comprising determining the total fat mass and / or android fat mass of the individual after administration, and administering the composition until the total fat mass and / or android fat mass of the individual is reduced by at least 3.5%.

44. The use of any one of claims 27-30, wherein the antibody or antigen-binding fragment that specifically binds GDF8 comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 360 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

368.

45. The use of any one of claims 27-30, wherein the antibody or antigen-binding fragment that specifically binds Activin A comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 553 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

537.

46. The use of any one of claims 27-30, wherein the amount of the Activin A inhibitor is 100% to 400% of the amount of the GDF8 inhibitor by weight.

47. The use of claim 46, wherein the amount of the Activin A inhibitor is 1.5 to 2.0 times the amount of the GDF8 inhibitor by weight.

48. The use of any one of claims 27-30, wherein the reduction in fat mass in the individual is a reduction in total fat mass as measured by DXA (dual-energy X-ray absorptiometry).

49. The use of any one of claims 27-30, wherein the reduction in fat mass in the individual is a reduction in male-type fat mass as measured by DXA (dual-energy X-ray absorptiometry).

50. The use of any one of claims 27-30, wherein the individual experiences an increase in muscle volume after administration.

51. The use of any one of claims 27-30, wherein the individual does not have a muscle wasting disorder or disease.

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